Method of constructing novel higher plant and method of promoting the growth of higher plant

ABSTRACT

The present invention provides a method of producing a higher plant having cytochrome c 6  in the thylakoid space of chloroplast, which is characterized in that it comprises introducing a gene encoding a fused protein formed by adding a signal peptide consisting of 50 to 80 amino acid residues to a cytochrome c 6  protein into the genome of a higher plant.

This application is a Division of co-pending application Ser. No.11/883,240, filed on Jul. 26, 2007, which is the national stageapplication of PCT International Application No. PCT/JP2006/302104,filed Feb. 1, 2006. The present application also claims the benefit ofpriority of Japanese Patent Application No. 2005-027012, filed Feb. 2,2005. The entire contents of all are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a method of producing a novel higherplant having cytochrome c₆ in the thylakoid space of chloroplast, and amethod of promoting the growth of a higher plant by allowing cytochromec₆ in the aforementioned thylakoid space of chloroplast or a method ofpromoting the ability of a higher plant to fix carbon.

BACKGROUND ART

As a technique of promoting the growth of a so-called higher plant suchas a land plant, a photosynthetic dark reaction (Calvin-Benson cycle),which involves the enhancement of the activity of enzyme such asribulosebisphosphate carboxylase, has previously been reported.Specifically, a technique of enlarging leaves by introducing a relatedenzyme gene into such a higher plant has been reported (Shigeoka et al.,Nature biotechnology, 19, 965-969 (2001)). However, it has beenextremely difficult to apply such techniques to various types of higherplants.

Cytochrome c₆ is an electron transfer protein in a photosynthetic lightreaction, and in general, it exists only in several types of algae(blue-green algae, etc.) It has been known that cytochrome c₆ hasexcellent electron-transferring ability (that is, itsoxidation-reduction potential is high) (FIG. 1). Thus, it has beenstrongly desired that a general-purpose technique of allowing cytochromec₆ to express and function in the chloroplast (more in detail, in thethylakoid space) of various types of higher plants, so as to improvephotosynthetic ability, be developed.

By the way, examples of a technique of allowing cytochrome c₆ to expressin a cell include those described in publications such as F. P.Molina-Heredia et al., Biochem. Biophys. Res. Commun., 243, 302-306(1998); T. Satoh et al., FEBS lett., 531, 543-547 (2002); R. Gupta etal., Nature, 417, 567-571 (2002); and D. R. Hickey et al., Gene, 105,73-81 (1991). However, in all of these techniques, host cells have notbeen those of higher plants, and cells of Escherichia coli, yeast, orblue-green algae, have been used just for simple purposes, such asmass-production of the aforementioned cytochrome c₆ or a proteinassociated therewith, or the function analysis thereof. Hence, suchtechniques have been included in the gene expression method, which hasbeen commonly carried out by persons skilled in the art.

There have been no reports regarding that cytochrome c₆ is allowed tofunction as an electron carrier in a photosynthetic light reaction inhigher plants, namely, regarding that cytochrome c₆ is successfullyallowed to exist in the thylakoid space of the chloroplast of a higherplant cell. Thus, such a technique has been considered extremelydifficult.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method ofproducing a novel higher plant having cytochrome c₆ in the thylakoidspace of chloroplast, and eventually, to provide a method of promotingthe growth of a higher plant, a method of promoting the synthesis of atleast one selected from the group consisting of ATP, NADPH, a starch anda protein, and a method of promoting carbon fixation.

The present inventor has conducted intensive studies directed towardsachieving the aforementioned object. As a result, the inventor has foundthat if a cytochrome c₆ protein gene, to which a specific signal peptidehas been added, is introduced into the genomic DNA of a higher plant soas to allow the gene to express therein, it becomes possible to transfercytochrome c₆ into (allow cytochrome c₆ to pass through) the chloroplastenvelope and thylakoid membrane of the above higher plant, which hadconventionally been considered to be impossible, thereby completing thepresent invention.

That is to say, the present invention has the following features (1) to(15).

(1) A method of producing a higher plant having cytochrome c₆ in thethylakoid space of chloroplast, which is characterized in that itcomprises introducing a gene encoding a fused protein formed by adding asignal peptide consisting of 50 to 80 amino acid residues to acytochrome c₆ protein into the genome of a higher plant.(2) A method of promoting the growth of a higher plant, which ischaracterized in that it comprises introducing a gene encoding a fusedprotein formed by adding a signal peptide consisting of 50 to 80 aminoacid residues to a cytochrome c₆ protein into the genome of a higherplant, so as to allow the gene to express therein, and allowingcytochrome c₆ to exist in the thylakoid space of chloroplast.(3) A method of promoting the synthesis of at least one selected fromthe group consisting of the ATP, NADPH, starch, and protein of a higherplant, which is characterized in that it comprises introducing a geneencoding a fused protein formed by adding a signal peptide consisting of50 to 80 amino acid residues to a cytochrome c₆ protein into the genomeof a higher plant, so as to allow the gene to express therein, andallowing cytochrome c₆ to exist in the thylakoid space of chloroplast.(4) A method of promoting carbon fixation by a higher plant, which ischaracterized in that it comprises introducing a gene encoding a fusedprotein formed by adding a signal peptide consisting of 50 to 80 aminoacid residues to a cytochrome c₆ protein into the genome of a higherplant, so as to allow the gene to express therein, and allowingcytochrome c₆ to exist in the thylakoid space of chloroplast.

In the method according to any one of (1) to (4) above, theabove-described fused protein may be the protein described in thefollowing (a), (b), (c), or (d):

(a) a protein having the amino acid sequence as shown in SEQ ID NO: 6;(b) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toan amino acid sequence portion corresponding to the above-describedsignal peptide in the amino acid sequence as shown in SEQ ID NO: 6, andwhich has ability to pass through the chloroplast envelope and thylakoidmembrane of a higher plant;(c) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toan amino acid sequence portion corresponding to the above-describedcytochrome c₆ protein in the amino acid sequence as shown in SEQ ID NO:6, and which has ability to transfer electrons; and(d) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toeach of an amino acid sequence portion corresponding to theabove-described signal peptide and an amino acid sequence portioncorresponding to the above-described cytochrome c₆ protein in the aminoacid sequence as shown in SEQ ID NO: 6, and which has ability to passthrough the chloroplast envelope and thylakoid membrane of a higherplant and ability to transfer electrons.

In addition, in the method according to any one of (1) to (4) above, theabove-described gene may be a gene comprising the DNA described in thefollowing (a) or (b):

(a) DNA having the nucleotide sequence as shown in SEQ ID NO: 5; and(b) DNA, which hybridizes with DNA having a nucleotide sequencecomplementary to the DNA having the nucleotide sequence as shown in SEQID NO: 5 under stringent conditions, and which encodes a protein havingability to pass through the chloroplast envelope and thylakoid membraneof a higher plant and ability to transfer electrons.(5) A fused protein, which is formed by adding a signal peptideconsisting of 50 to 80 amino acid residues to a cytochrome c₆ protein.

The fused protein according to (5) above may be the protein described inthe following (a), (b), (c), or (d):

(a) a protein having the amino acid sequence as shown in SEQ ID NO: 6;(b) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toan amino acid sequence portion corresponding to the above-describedsignal peptide in the amino acid sequence as shown in SEQ ID NO: 6, andwhich has ability to pass through the chloroplast envelope and thylakoidmembrane of a higher plant;(c) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toan amino acid sequence portion corresponding to the above-describedcytochrome c₆ protein in the amino acid sequence as shown in SEQ ID NO:6, and which has ability to transfer electrons; and(d) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toeach of an amino acid sequence portion corresponding to theabove-described signal peptide and an amino acid sequence portioncorresponding to the above-described cytochrome c₆ protein in the aminoacid sequence as shown in SEQ ID NO: 6, and which has ability to passthrough the chloroplast envelope and thylakoid membrane of a higherplant and ability to transfer electrons.(6) A gene, which encodes the fused protein according to (5) above.(7) A gene, which comprises the DNA described in the following (a) or(b):(a) DNA having the nucleotide sequence as shown in SEQ ID NO: 5; and(b) DNA, which hybridizes with DNA having a nucleotide sequencecomplementary to the DNA having the nucleotide sequence as shown in SEQID NO: 5 under stringent conditions, and which encodes a protein havingability to pass through the chloroplast envelope and thylakoid membraneof a higher plant and ability to transfer electrons.(8) A recombinant vector, which comprises the gene according to (6) or(7) above.(9) A transformant, which is obtained by introducing the recombinantvector according to (8) above into a host.

The transformant according to (9) above may be a transformant whereinthe host is a microorganism belonging to genus Agrobacterium.

(10) A transgenic higher plant, which is obtained by introducing thegene according to (6) or (7) above into the plant genome thereof.

The transgenic higher plant according to (10) above preferably hascytochrome c₆ in the thylakoid space of chloroplast in a plant cellthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the electron transport system in photosynthesisof a chloroplast thylakoid membrane.

FIG. 2 is a schematic diagram showing introduction of a cytochrome c₆gene into a plant via Agrobacterium and the expression manner ofcytochrome c₆ in the plant in the present invention.

FIG. 3 is a view showing a difference in the electron transfer systembetween cyanobacteria and algae, and higher plants.

FIG. 4 is a view showing the nucleotide sequence (SEQ ID NO: 13) of thecDNA of Porphyra yezoensis cytochrome c₆ and a putative amino acidsequence (SEQ ID NO: 14).

FIG. 5 is a view showing the results obtained by amplifying the matureprotein region of a Porphyra yezoensis cytochrome c₆ gene by PCR.

FIG. 6 is a view showing a pBluescript II SK+/− vector used inconstruction of a cytochrome c₆ gene to be introduced into plants.

FIG. 7 is a view showing the nucleotide sequence (SEQ ID NO: 15) of agene that is formed by fusing a signal peptide of plastocyanin derivedfrom Arabidopsis thaliana with a mature protein region of cytochrome c₆derived from Porphyra yezoensis, and a putative amino acid sequence (SEQID NO: 16).

FIG. 8 is a schematic diagram showing a method of transformingAgrobacterium tumefaciens by triparental mating.

FIG. 9 is a view showing the results obtained by performingelectrophoresis on a cytochrome c₆ gene introduced into a plant, whichhas been specified by PCR. (A) shows the results obtained using genomicDNA as a template, and (B) shows the results obtained using total RNA asa template.

FIG. 10 is a view showing the results obtained by specifying byelectrophoresis cytochrome c₆ that has been allowed to express in aplant. (A) shows the results obtained by CBB staining, and (B) shows theresults of Western blotting, wherein cytochrome c₆ has been allowed toreact with a cytochrome c₆ antibody.

FIG. 11 is a view showing a change in the height of a wild type (◯) andin the height of transgenic A. thaliana (), which has been observedwith the course of growing days. Each value in the graph indicatesaverage±S.D. (standard deviation) (n=20). The asterisk (*) indicates avalue having a significant difference at 5% significance level.

FIG. 12 is a view showing a change in the root length of a wild type (◯)and in the root length of transgenic A. thaliana (), which has beenobserved with the course of growing days. Each value in the graphindicates average±S.D. (standard deviation) (n=20). The asterisk (*)indicates a value having a significant difference at 5% significancelevel.

FIG. 13 is a view showing a change in the leaf size of a wild type (◯)and in the leaf size of transgenic A. thaliana (), which has beenobserved with the course of growing days. Each value in the graphindicates average±S.D. (standard deviation) (n=20). The asterisk (*)indicates a value having a significant difference at 5% significancelevel.

FIG. 14 is a view showing a change in the chlorophyll content of a wildtype and in the chlorophyll content of transgenic A. thaliana, obtained3 hours after light irradiation, which has been observed with the courseof growing days. Each value in the graph indicates average±S.D.(standard deviation) (n=8). The asterisk (*) indicates a value having asignificant difference at 5% significance level.

FIG. 15 is a view showing a change in the ATP content of a wild type andin the ATP content of transgenic A. thaliana, obtained 3 hours afterlight irradiation, which has been observed with the course of growingdays. Each value in the graph indicates average±S.D. (standarddeviation) (n=8). The asterisk (*) indicates a value having asignificant difference at 5% significance level.

FIG. 16 is a view showing a change in the NADPH content of a wild typeand in the NADPH content of transgenic A. thaliana, obtained 3 hoursafter light irradiation, which has been observed with the course ofgrowing days. Each value in the graph indicates average±S.D. (standarddeviation) (n=8). The asterisk (*) indicates a value having asignificant difference at 5% significance level.

FIG. 17 is a view showing a change in the carbon dioxide-assimilatingability of a wild type and in the carbon dioxide-assimilating ability oftransgenic A. thaliana, obtained 3 hours after light irradiation, whichhas been observed with the course of growing days. Each value in thegraph indicates average±S.D. (standard deviation) (n=10). The asterisk(*) indicates a value having a significant difference at 5% significancelevel.

FIG. 18 is a view showing a change in the starch amount contained in awild type and in the starch amount contained in transgenic A. thaliana,obtained 3 hours after light irradiation, which has been observed withthe course of growing days. Each value in the graph indicatesaverage±S.D. (standard deviation) (n=10). The asterisk (*) indicates avalue having a significant difference at 5% significance level.

FIG. 19 is a view showing a change in the protein amount contained in awild type and in the protein amount contained in transgenic A. thaliana,obtained 3 hours after light irradiation, which has been observed withthe course of growing days. Each value in the graph indicatesaverage±S.D. (standard deviation) (n=8). The asterisk (*) indicates avalue having a significant difference at 5% significance level.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail below. However, thefollowing descriptions are not intended to limit the scope of thepresent invention. Even except for the following examples, changes andmodifications may be made without departing from the spirit of theinvention.

The present specification includes all of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.2005-27012, which is a priority document of the present application. Inaddition, all prior art publications, patent publications, patentapplications, and other patent documents cited herein are incorporatedherein by reference in their entirety.

1. Summary of the present Invention

The present invention relates to a method of promoting the growth of ahigher plant, which comprises introducing a gene encoding a fusedprotein consisting of cytochrome c₆ and a signal peptide into a higherplant, so as to allow cytochrome c₆ acting as an electron carrier in aphotosynthetic light reaction to express and function in the thylakoidspace of chloroplast of such a higher plant cell (FIG. 2). By thepresent invention, the synthesis of ATP, NADPH, starch, and protein ispromoted in a higher plant cell, and a photosynthetic dark reaction(carbon fixation reaction) of converting carbon dioxide (CO₂) tocarbohydrate can be thereby promoted. The present invention includessuch a method of promoting the synthesis of ATP or carbon fixation in ahigher plant, and also includes a method of producing a higher plant,which involves promotion of the growth thereof, promotion of thesynthesis of ATP or the like, and promotion of carbon fixation.

In general, in a photosynthetic light reaction (photosyntheticelectron-transfer reaction), chlorophyll electrons acquire energy fromsunlight, and they move one another through an electron transport chainin the thylakoid space (in the thylakoid membrane). At that time,chlorophyll acquires electrons from water and discharges oxygen (O₂). Atthe same time, with such electron transfer reaction, H⁺ is pumped outthrough the thylakoid membrane, and ATP is synthesized in the stroma ofchloroplast due to the thus generated proton motive force. Thereafter,at the final stage of a series of reactions, NADP⁺ accepts high energyelectrons (as well as H⁺), so that NADPH can be synthesized.

In contrast, in a photosynthetic dark reaction (carbon fixationreaction), ATP and NADPH, which have been synthesized by aphotosynthetic light reaction, function as an energy source and areducing power, respectively. As a result, CO₂ is converted tocarbohydrate in chloroplast stroma and cytoplast. By this carbonfixation reaction, sucrose is synthesized in plant leaves and the like.Sucrose is transported to other tissues, and it is used as a syntheticmaterial for various organic molecules, and in particular, it is used asan energy source for the growth of the plant itself.

In general, an electron carrier in a photosynthetic light reaction in ahigher plant (an electron carrier protein) is considered to beplastocyanin (PC). However, it has been known that cytochrome c₆ actingas an electron carrier in algae is superior to plastocyanin (PC) interms of electron-transferring ability (high oxidation-reductionpotential) (FIG. 3). Thus, various attempts have previously been made toallow such cytochrome c₆ to express and function in a higher plant, soas to promote a photosynthetic light reaction and a photosynthetic darkreaction. However, as a matter of fact, it has been extremely difficultto carry out such attempts successfully. In fact, there have been nosuccessful examples to date.

In order that a protein that has been allowed to express in a cell of ahigher plant based on the genome information thereof exhibits a functionas an electron carrier in a photosynthetic light reaction, it isessential for the protein to exist in the thylakoid space thereof. Inorder that the protein exists in the thylakoid space, in general, itmust pass through two types of membranes, a “chloroplast envelope” and a“thylakoid membrane.” This point has been considered to be a main causefor extreme difficulty in expression of the function of cytochrome c₆ ina higher plant. Hence, when a cytochrome c₆ gene has been introducedinto the genome of a higher plant via genetic recombination, the presentinventor had previously constructed a signal sequence-added gene, suchthat the cytochrome c₆ protein could be expressed in the form of a fusedprotein obtained by adding a specific signal peptide (a signal peptidehaving a specific length or a specific amino acid sequence) to the abovecytochrome c₆ protein. Thereafter, the inventor has introduced the geneinto the higher plant. As a result, the presence of the cytochrome c₆protein (without the signal peptide) was observed in the thylakoid spaceof a plant body produced by the above gene introduction. Thus, it wasdemonstrated that the expressed fused protein is able to pass throughthe aforementioned two types of membranes due to the action of thesignal peptide thereof, thereby completing the present invention.

(1) Studies Regarding PYC6 Gene Introduction System

To date, it has been known that the oxidation-reduction potential ofPYC6 is high. In addition, the genetic sequence of PYC6 has beenclarified. In the present invention, the PYC6 gene was first ligated toa binary vector, so as to prepare Agrobacterium used in transformation.Arabidopsis thaliana was disseminated and was then subjected to alow-temperature treatment. Thereafter, it was allowed to grown underlong day conditions. At the stage of formation of flower buds,Arabidopsis thaliana was transformed by infiltration under reducedpressure. Subsequently, in order to analyze gene introduction andexpression in the obtained transformant, using genomic DNA and RNAextracted from the plant body as templates, PCR and RT-PCR were carriedout, respectively. As a result, amplification of the PYC6 gene wasobserved, and it was confirmed using a DNA sequencer that the nucleotidesequence thereof is the same as that of the PYC6 gene. Subsequently, inorder to confirm expression of the PYC6 gene observed in thetransformant as a protein, a chloroplast protein fraction was extractedfrom the plant body, and it was then electrophoresed, followed byperforming Western blotting. As a result, expression of the PYC6 proteinwas confirmed. In addition, the N-terminal amino acid sequence of thePYC6 protein in the obtained transformant was analyzed. As a result, itwas found that the obtained N-terminal amino acid sequence was identicalto the amino acid sequence of the introduced PYC6 (PIR accession No.JC5849). From these results, it was found that cyt c₆ (PYC6) derivedfrom algae was successfully allowed to express in A. thaliana that was ahigher plant.

(2) Influence of PYC6 Introduction upon Plant Body

The growth of a PYC6-introduced plant was observed. As a result, asshown in the examples as given later, the size of a leaf thereof wasapproximately 1.2 to 1.3 times greater than that of a wild type (WT),and the height thereof was approximately 1.5 times greater than that ofthe wild type (WT). At the initial growth stage, the PYC6-introducedplant had a growth rate that was higher than that of WT. Moreover, as aresult of the measurement of the chlorophyll amount contained in thePYC6-introduced plant, it was found that the chlorophyll amountcontained in the PYC6-introduced plant was approximately 1.1 to 1.2times larger than that of WT. It is considered that such a phenomenonoccurred in the plant body as a result of the PYC6 introduction wasbrought on as a result of activation of the entire photosyntheticreaction using an energetic substance as a final product of a lightreaction. Thus, the amount of ATP contained in the PYC6-introduced plantwas measured by the luciferin-luciferase method. As a result, it wasfound that the ATP amount contained in the PYC6-introduced plant wasapproximately 1.7 times larger than that of WT. It was considered thatthis was because electron transfer in a light reaction was activated byP. yezoensis cyt c₆ that functioned as a novel electron carrier, as wellas plastocyanin that functioned in A. thaliana, and the amount of ATP asa final product was thereby increased, and the growth of the plant as awhole was also thereby activated.

When the above fused protein passes through the aforementioned two typesof membranes, a part of a signal peptide is used when the protein passesthrough the first membrane (chloroplast envelope), and the remainingpart is used when it passes through the second membrane (thylakoidmembrane). Each of the two parts was dissociated from the protein afteruse, and finally, the above protein is transferred into the thylakoidspace in the form of not having such a signal peptide. It is consideredthat the thus transferred PYC6 protein binds to heme (heme c) in thethylakoid space, followed by folding, so that it becomes cytochrome c₆that is able to function as an electron carrier.

2. Method of Producing Novel Higher Plant

As described above, the production method of the present invention is amethod of producing a novel higher plant having cytochrome c₆ in thethylakoid space of chloroplast, which is characterized in that itcomprises introducing a gene encoding a fused protein formed by adding asignal peptide consisting of 50 to 80 amino acid residues to acytochrome c₆ protein into the genome of a higher plant.

(1) Higher Plant to be Produced

The type of a higher plant that can be used in the production method ofthe present invention, namely, the type of a higher plant that can betransformed such that it has cytochrome c₆ in the thylakoid space ofchloroplast, is not limited. For example, the following plants can beused.

The plant used in the present invention includes all of a plant body asa whole, a plant organ (for example, a leaf, a petal, a pedicle, a root,a seed, etc.), a plant tissue (for example, epidermis, phloem,parenchyma, xylem, vascular bundle, palisade tissue, spongy tissue,etc.), and a cultured plant cell (including cultured tissues such ascallus). Plants used in transformation include C3, C4 and CAM plants,and all of their intermediate plants. Examples of such a plant includeplants belonging to Brassicaceae, Solanaceae, Gramineae, Leguminosae,Chenopodiaceae, Rosaceae, Asteraceae, Liliaceae, Caryophyllaceae,Cucurbitaceae, Convolvulaceae, Amaranthaceae, Bromeliaceae, Cactaceae,and Aloeaceae (including fruits, vegetables, flowers and ornamentalplants, and the like) (see below). However, examples are not limitedthereto.

[C3 Plants]

-   -   Brassicaceae: Arabidopsis thaliana, Raphanus, etc.    -   Solanaceae: Nicotiana tabacum, Solanum, etc.    -   Gramineae: Oryza sativa, Zea mays, Triticun, etc.    -   Leguminosae: Glycine max, Pisum, etc.    -   Chenopodiaceae: Spinacia, etc.    -   Rosaceae: Prunus, Rosa, etc.    -   Asteraceae: Erigeron, Taraxacun, etc.    -   Cucurbitaceae: Cucurdida, Cucumis, etc.    -   Convolvulaceae: Ipomea, etc.    -   Orchidaceae: Poneorchis, etc.

[C4 Plants]

-   -   Gramineae: Zea mays, Saccharum officinarum, Setaria itarica,        etc.    -   Amaranthaceae: Amaranthaceae, etc.

[CAM Plants]

-   -   Bromeliaceae: Ananas comosus, etc.    -   Cactaceae: Lophosphora difusa, Opuntia spp., etc.    -   Aloeaceae: Aloe arborescens, Aloe vera, etc.

[C3-C4 Intermediate Plants]

-   -   Aizoaceae: Mollugo verticillata, etc.    -   Gramineae: Panicum milioides, etc.

Among others, as an effect of the production method of the presentinvention, if taking into consideration the achievement of a planthaving a high growth-promoting effect, highly marketable plants areadvantageous for the present invention. Specifically, preferred examplesof such a highly marketable plant include foliage plants (spinach,cabbage, etc.), flower plants (rose, phalaenopsis, etc.), pedicle plants(potato, lotus root, etc.), root plants (burdock, Japanese radish,etc.), grains (rice, wheat, etc.), fruits (pineapple, grape, etc.),ornamental plants (Pinus, maple, etc.), and woods (Cryptomeria japonica,Chamaecyparis obtuse, etc.).

(2) Fused Protein

It is important for the production method of the present invention tointroduce a gene encoding a specific fused protein into the genome of ahigher plant, or to use a fused protein formed by adding a signalpeptide having a certain length to a cytochrome c₆ protein.

It is important that the above fused protein has a signal peptide havinga length of 50 to 80 amino acid residues. Thus, the fused protein has asignal peptide having a sufficient length, so that it makes thecytochrome c₆ protein to pass through both the chloroplast envelope andthe thylakoid membrane. The sequence of such a signal peptide can bedetermined by gene cloning through higher plants.

In general, the signal peptide in the above fused protein is preferablyadded to the N-terminus of the cytochrome c₆ protein.

In the present invention, the above fused protein is preferably theprotein described in the following (a), (b), (c), or (d), for example:

(a) a protein having the amino acid sequence as shown in SEQ ID NO: 6;(b) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toan amino acid sequence portion corresponding to the above-describedsignal peptide in the amino acid sequence as shown in SEQ ID NO: 6, andwhich has ability to pass through the chloroplast envelope and thylakoidmembrane of a higher plant;(c) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toan amino acid sequence portion corresponding to the above-describedcytochrome c₆ protein in the amino acid sequence as shown in SEQ ID NO:6, and which has ability to transfer electrons; and(d) a protein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toeach of an amino acid sequence portion corresponding to theabove-described signal peptide and an amino acid sequence portioncorresponding to the above-described cytochrome c₆ protein in the aminoacid sequence as shown in SEQ ID NO: 6, and which has ability to passthrough the chloroplast envelope and thylakoid membrane of a higherplant and ability to transfer electrons.

As shown in SEQ ID NO: 6, the protein described in (a) above consists oftotal 157 amino acids, and it is formed by adding a signal peptidesequence consisting of 72 amino acids (SEQ ID NO: 4) to the N-terminalside of a cytochrome c₆ protein sequence consisting of 85 amino acids(SEQ ID NO: 2). It may also be a fused protein having the amino acidsequence as shown in SEQ ID NO: 6. However, such a signal peptide is notlimited to the above signal peptide having 72 amino acids. The origin ofthe cytochrome c₆ protein is not particularly limited herein, andPorphyra yezoensis, blue-green algae, brown algae, Bacillariophyta,Chlorophyceae, and the like can be used. The cytochrome c₆ protein usedherein is preferably derived from P. yezoensis. The amino acid sequenceof the cytochrome c₆ protein derived from P. yezoensis has been known(PIR accession No. JC5849). It can be obtained by database searching.

The type of the protein described in (b) above is not limited, as longas it has an amino acid sequence comprising a deletion, substitution, oraddition of one or several amino acids (for example approximately 1 to10 amino acids, and preferably approximately 1 to 5 amino acids) withrespect to the amino acid sequence portion consisting of 72 amino acids(SEQ ID NO: 4) corresponding to the above-described signal peptide inthe total amino acid sequence that constitutes the protein described in(a) above, and which has ability to pass through the chloroplastenvelope and thylakoid membrane of a higher plant.

It is important for the protein described in (b) above to have abilityto pass through the chloroplast envelope and thylakoid membrane of ahigher plant, as with the protein described in (a) above. Accordingly,it is preferable that an amino acid sequence portion that is consideredto be important for passing through the above chloroplast envelope orthylakoid membrane, such as amino acids from position 53 to position 72,be not mutated or substituted from the amino acid sequence of theprotein described in (a) above.

The type of the protein described in (c) above is not limited, as longas it has an amino acid sequence comprising a deletion, substitution, oraddition of one or several amino acids (for example approximately 1 to10 amino acids, and preferably approximately 1 to 5 amino acids) withrespect to the amino acid sequence portion consisting of 85 amino acids(SEQ ID NO: 6) corresponding to the above-described cytochrome c₆protein in the total amino acid sequence that constitutes the proteindescribed in (a) above, and which has ability to transfer electrons (ina photosynthetic light reaction).

Herein, the above term “protein having ability to transfer electrons” isused in the present invention to mean a protein having an amino acidsequence portion obtained by eliminating an amino acid sequence portioncorresponding to the above-described signal peptide from the total aminoacid sequence that constitutes the protein described in (c) above,wherein the above-described protein has electron-transferring abilityafter it has bound to heme (heme c).

As in the case of the cytochrome c₆ protein, it is important for theprotein described in (c) above to have electron-transferring ability.Accordingly, it is preferable that an amino acid sequence portion thatis considered to be important for functioning as an electron carrier inthe photosynthetic light reaction of a higher plant, such as amino acidsfrom position 14 to position 18 and amino acids from position 47 to 60,be not mutated or substituted from the amino acid sequence of theprotein described in (a) above.

The type of the protein described in (d) above is not limited, as longas it has an amino acid sequence comprising a deletion, substitution, oraddition of one or several amino acids (for example approximately 1 to10 amino acids, and preferably approximately 1 to 5 amino acids) withrespect to the amino acid sequence portion consisting of 72 amino acidscorresponding to the above-described signal peptide (SEQ ID NO: 4) andalso has an amino acid sequence comprising a deletion, substitution, oraddition of one or several amino acids (for example approximately 1 to10 amino acids, and preferably approximately 1 to 5 amino acids) withrespect to the amino acid sequence portion consisting of 85 amino acidscorresponding to the above-described cytochrome c₆ protein (SEQ ID NO:2), in the total amino acid sequence that constitutes the proteindescribed in (a) above, and which has ability to pass through thechloroplast envelope and thylakoid membrane of a higher plant andability to transfer electrons (in a photosynthetic light reaction).

Herein, the above term “protein having ability to transfer electrons”has the same meaning as that in the case of the protein described in (c)above.

It is important for the protein described in (d) above to have abilityto pass through the chloroplast envelope and thylakoid membrane of ahigher plant as with the protein described in (a) above, and also tohave electron-transferring ability as with the cytochrome c₆ protein.Accordingly, it is preferable that an amino acid sequence portion thatis considered to be important for passing through the above chloroplastenvelope or thylakoid membrane, such as amino acids from position 53 toposition 72, or an amino acid sequence portion that is considered to beimportant for functioning as an electron carrier in the photosyntheticlight reaction of a higher plant, such as amino acids from position 14to position 18 and amino acids from position 47 to 60, be not mutated orsubstituted from the amino acid sequence of the protein described in (a)above.

The presence or absence of the electron-transferring ability of theproteins described in (c) and (d) above, or such electron-transferringability if the above proteins have such ability, can be confirmed ormeasured by three-dimensional structure analysis and the measurement ofoxidation-reduction potential. It is to be noted that theoxidation-reduction potential of cytochrome c₆ is between approximately350 and 370 mV.

A polynucleotide encoding an amino acid sequence comprising a deletion,insertion, or addition of one or several amino acids with respect to theamino acid sequence as shown in SEQ ID NO: 6 can be prepared accordingto methods such as site-directed mutagenesis described in “MolecularCloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989));“Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997);Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92; and Kunkel (1988)Method. Enzymol. 85: 2763-6; etc. Such a gene encoding a mutant aminoacid sequence, such as an amino acid sequence comprising a deletion,substitution, addition, etc. of amino acids, can be produced by knownmethods such as the Kunkel method or the Gapped duplex method, using amutation introduction kit that utilizes site-directed mutagenesis, suchas QuikChange™ Site-Directed Mutagenesis Kit (manufactured byStratagene), GeneTailor™ Site-Directed Mutagenesis System (manufacturedby Invitrogen), or TaKaRa Site-Directed Mutagenesis System (Mutan-K,Mutan-Super Express Km, etc.: manufactured by TAKARA BIO INC.).

(3) Gene

In the production method of the present invention, it is important thata gene to be introduced into the genome of a higher plant is a geneencoding the aforementioned fused protein.

In the present invention, the above gene preferably comprises the DNAdescribed in the following (a) or (b), for example. It is to be notedthat both the DNAs described in the following (a) and (b) are thestructural genes of the aforementioned fused protein (namely, a geneformed by ligating a gene encoding a signal peptide to the structuralgene of cytochrome c₆ or a protein having electron-transferring abilityequivalent to that of the cytochrome c₆). A gene comprising such DNAsmay consist of only such DNAs. Otherwise, it may comprise such DNAs andother known nucleotide sequences necessary for expression of thestructural gene of the above fused protein (a transcriptional promoter,an SD sequence, a Kozak sequence, a terminator, etc.). Thus, the type ofa gene comprising such DNAs is not limited. The nucleotide sequenceencoding cytochrome c₆ has been made public to DNA Data Bank of Japan(DDBJ), and thus it has been known (Accession number: AB40818).

(a) DNA having the nucleotide sequence as shown in SEQ ID NO: 5

(b) DNA, which hybridizes with DNA having a nucleotide sequencecomplementary to the DNA having the nucleotide sequence as shown in SEQID NO: 5 under stringent conditions, and which encodes a protein havingability to pass through the chloroplast envelope and thylakoid membraneof a higher plant and ability to transfer electrons

The DNA described in (b) above can be obtained by performing a knownhybridization method such as colony hybridization, plaque hybridization,or Southern blotting, using the DNA described in (a) above, DNA having anucleotide sequence complementary thereto, or a fragment thereof, as aprobe, and then utilizing a cDNA library or a genome library. As such alibrary, a library produced by a known method may be used. Otherwise, acommercially available cDNA library or genome library may also be used.Thus, the type of a library used herein is not limited.

In addition, both the nucleotide sequence encoding the signal peptideportion (SEQ ID NO: 3) and the nucleotide sequence encoding cytochromec₆ (SEQ ID NO: 1), or either the nucleotide sequence as shown in SEQ IDNO: 3 or the nucleotide sequence as shown in SEQ ID NO: 1, may comprisea mutation.

For detailed procedures of hybridization methods, please refer toMolecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring HarborLaboratory Press (1989)) or other publications, as appropriate.

The term “stringent conditions” regarding hybridization methods is usedto mean conditions that are applied during washing after hybridization.Such stringent conditions are that the salt concentration in a buffer isbetween 15 and 750 mM, and preferably between 15 and 150 mM, and thatthe temperature is between 25° C. and 65° C., and preferably between 45°C. and 55° C. Specific examples are conditions consisting of 50 mM and50° C. Moreover, in addition to conditions such as a salt concentrationand temperature, taking into consideration various conditions such as aprobe concentration, a probe length, or a reaction time, conditionsnecessary for obtaining the DNA described in (b) above can bedetermined, as appropriate.

Furthermore, the DNA described in (b) above can be obtained by producinga probe using a suitable fragment according to a method known to personsskilled in the art, then performing a known hybridization method such ascolony hybridization, plaque hybridization, or Southern blotting, usingthe above probe, and then utilizing a cDNA library and a genome library.Examples of stringent conditions applied in the above hybridizationinclude 1×SSC to 2×SSC, 0.1% to 0.5% SDS, and 30° C. to 80° C. Morespecifically, after completion of pre-hybridization at 60° C. to 68° C.for 30 minutes or more, a probe is added to the reaction product, andthe mixture is retained at 68° C. for 1 hour or more, so as to form ahybrid. Thereafter, the hybrid is washed with 2×SSC in 0.1% SDS once ortwice at room temperature for 5 to 15 minutes.

DNA used in hybridization has a nucleotide sequence showing homology ofat least 70%, more preferably 80% or more, further more preferably 90%or more, and particularly preferably 95% or more, with the nucleotidesequence of the DNA described in (a) above.

It is important that the DNA described in (b) above encodes a proteinhaving ability to pass through the chloroplast envelope and thylakoidmembrane of a higher plant and ability to transfer electrons.Accordingly, taking into consideration the amino acid sequence obtainedafter translation, the above DNA preferably has a nucleotide sequence,regarding which an amino acid sequence portion that is considered to beimportant for passing through the above chloroplast envelope orthylakoid membrane, such as amino acids from position 53 to position 72,or an amino acid sequence portion that is considered to be important forfunctioning as an electron carrier in the photosynthetic light reactionof a higher plant, such as amino acids from position 14 to position 18and amino acids from position 47 to 60, be not mutated or substitutedfrom the amino acid sequence of the protein described in (a) above.

Preferred examples of the DNA described in (b) above include: DNA thathas been mutated, such that the amino acid obtained by substituting thenucleotide at position 103 “T (thymine)” with “A (adenine)” of the DNAdescribed in (a) above, followed by translation, is changed from “Ser(serine)” to “Thr (threonine);” DNA that has been mutated, such that thetranslated amino acid is not changed although the nucleotide at position216 “C (cytosine)” has been substituted with “T (thymine);” and DNA thathas been mutated by the combined use of such nucleotide substitutions.

In the present invention, the above gene is allowed to express in ahigher plant. Thus, it is necessary that the above gene comprise DNAwherein codons corresponding to amino acids are those commonly used inplants after transcription (preferably, frequently-used codons).

(4) Gene Introduction Method

The type of a method of producing a novel higher plant (a transgenichigher plant) by introducing a gene encoding the aforementioned fusedprotein (a gene of interest) into the genome of a higher plant withoutchanging other traits is not limited. Known genetic recombinationtechniques such as the Agrobacterium method, the electroporation method,or the particle gun method, can be arbitrarily adopted. For example, avacuum infiltration method using bacteria of genus Agrobacterium (crowngall bacteria), into which a binary vector comprising a gene of interesthas been introduced, is preferably applied, but examples are not limitedthereto. The details will be described below.

(i) Recombinant Vector

The type of a recombinant vector comprising a gene encoding theaforementioned fused protein is not limited. A vector that can be usedas the aforementioned binary vector is preferable. Specifically, arecombinant vector constructed by inserting a gene of interest to beincorporated into plant genome (instead of a GUS gene) into a T-DNAregion of a vector having the T-DNA region that is cut out by the actionof a Vir region gene, is preferable. Preferred examples of a vectorhaving the above T-DNA region include a pBI121 vector (manufactured byClontech; the vector having a kanamycin resistance gene as a selectivemarker and comprising a GUS gene downstream of a 35S promoter), and apBI101 vector (manufactured by Clontech; the vector having a kanamycinresistance gene as a selective marker and comprising a GUS gene).

In addition, a recombinant vector that is constructed by inserting agene of interest into a known expression vector suitable for a host cellis also included in the recombinant vector of the present invention.However, this vector cannot be used for the vacuum infiltration method.As necessary, a transcriptional promoter, an SD sequence (in a casewhere a host is a prokaryocyte), or a Kozak sequence (in a case where ahost is a eukaryote) may be added upstream of the above gene by PCR orthe like. In addition, a terminator may be added downstream thereof byPCR or the like. Various elements necessary for expression of a fusedprotein, such as the aforementioned transcriptional promoter, may becomprised in a gene of interest. If such elements are originallycomprised in an expression vector, they may also be used. Thus, theorigins of such necessary elements are not limited. Such a recombinantvector can be used in mass production of the aforementioned fusedprotein, etc.

In order to produce various types of recombinant vectors, known geneticrecombination techniques and conditions, such as a method usingrestriction enzymes or a method using topoisomerase, can be adopted andused, as appropriate.

(ii) Transformant Produced by Introduction of Recombinant Vector

The type of a host, into which a recombinant vector is introduced, isnot limited. When the vacuum infiltration method is carried out at thefinal stage, bacteria of genus Agrobacterium (Agrobacterium tumefaciens,etc.) are used as a host, into which a binary vector is introduced. Whensuch bacteria of genus Agrobacterium are used, a previously-transformedcell mass (a transformant of bacteria of genus Agrobacterium), which isproduced by destroying or eliminating a cancer gene in the T-DNA regionon a Ti plasmid, originally owned by the above bacteria, is generallyused. In another case, when an expression vector is introduced as arecombinant vector, known hosts such as Escherichia coli, Bacillussubtilis, yeast, mold, or various types of animal cells including humancells or mouse cells, can be used.

The type of a method of transforming a host is not limited. While takinginto consideration the combination of a host and a recombinant vector, asuitable method can be appropriately selected from known methods, and itcan be applied. Preferred examples of such a transformation methodinclude electroporation, lipofection, the heat shock method, the PEGmethod, the calcium phosphate method, and the DEAE dextran method.

With respect to the obtained transformant, the actually used host may beeither identical to or different from a host with the codon type of anEBNA1 mutant gene contained in the recombinant vector. Thus, it is notlimited.

(iii) Transformation of Higher Plant and Transgenic Higher Plant

The type of a method of introducing a gene of interest into the genomeof a higher plant to produce a transgenic higher plant is not limited.The aforementioned vacuum infiltration method is preferable. Inaddition, it is preferable that a plant body to be transformed be in theform of an adult plant or a callus.

Specifically, a method of transforming a higher plant via the vacuuminfiltration method comprises: (a) infecting the lamina of a higherplant with transgenic bacteria (a transformant of bacteria of genusAgrobacterium) that contain a recombinant vector (a binary vector), intowhich a gene encoding the aforementioned fused protein has beeninserted; (b) culturing the lamina in a selective medium that containsantibiotics such as kanamycin; and (c) forming an adventitious budcallus and then allowing it to grow, so as to obtain a transgenic higherplant. When such a vacuum infiltration method is applied, all means andtreatment conditions applied in each treating step are not limited, andthey may be appropriately selected from a known range.

A gene encoding the aforementioned fused protein has been introducedinto the genome of the thus obtained transgenic higher plant. A fusedprotein allowed to express based on the above gene has theaforementioned signal peptide, and it has ability to pass through thechloroplast envelope and thylakoid membrane of a plant cell.Accordingly, the obtained transgenic higher plant has cytochrome c₆(preferably cytochrome c₆ acting as an electron carrier in aphotosynthetic light reaction) in the thylakoid space thereof.

It is considered that not only plastocyanin (PC) but also cytochrome c₆functions as an electron carrier in a photosynthetic light reaction inthe obtained transgenic higher plant, and promotion of the growth of theplant is thereby effectively achieved. However, the size (height, rootlength, leaf size, etc.) of the adult body of such a transgenic higherplant is not limited. The size of such a transgenic higher plant is forexample 1.1 times or more, preferably 1.2 times or more, and morepreferably 1.5 times or more (for example, approximately 1.9 times)greater than that of a wild-type higher plant.

In addition, such a transgenic higher plant has the effect of increasingthe numbers of various types of molecules in each cell, as well as theeffect of promoting the growth thereof. For example, the quantities ofchlorophyll molecules are not limited, but they are for example 1.1times or more, preferably 1.2 times or more, and more preferably 1.5times or more (for example, approximately 1.6 times) larger than thoseof a wild-type higher plant. Thus, the transgenic higher plant hasphotosynthetic ability that has been further improved.

Moreover, since the obtained transgenic higher plant has aphotosynthetic efficiency that has been improved, the synthetic amountof ATP as a product thereof is for example 1.1 times or more, preferably1.2 times or more, and more preferably 1.5 times or more (for example,approximately 1.7 times) larger than that of a wild-type higher plant,but such a synthetic amount is not limited thereto. Likewise, thesynthetic amount of NADPH is for example 1.1 times or more, preferably1.2 times or more, and more preferably 1.5 times or more (for example,approximately 1.7 times) larger than that of a wild-type higher plant,but such a synthetic amount is not limited thereto.

Furthermore, in the obtained transgenic higher plant, as the syntheticamount of ATP and that of NADPH increase, the efficiency of aphotosynthetic dark reaction, in which such ATP and NADPH function as anenergy source or a reduction power, is also improved. For example, thetransgenic higher plant has carbon fixation ability to convert carbondioxide (CO₂) to carbohydrate that is for example 1.1 times or more,preferably 1.2 times or more, and more preferably 1.4 times or moregreater than that of a wild-type higher plant.

Still further, as a result that the efficiency of a photosynthetic lightreaction or a photosynthetic dark reaction has been promoted in theobtained transgenic higher plant, the efficiency of protein synthesishas also been improved. For example, the transgenic higher plant has aprotein content that is for example 1.1 times or more, preferably 1.2times or more, and more preferably 1.5 times or more larger than that ofa wild-type higher plant.

Still further, sulfuric acid and nitric acid metabolism, the synthesisof various types of amino acids, the synthetic amount of lipid, and anincrease in pigment and flowers, etc. can also be anticipated.

The obtained transgenic higher plant has preferably at least one of,more preferably two or more of, and further more preferably all of,various types of the aforementioned effects.

3. Method of Promoting Growth of Higher Plant, Method of PromotingSynthesis of ATP, NADPH, Starch and Protein, and Method of PromotingCarbon Fixation

As described above, the obtained transgenic higher plant has (i) theeffect of promoting the growth thereof, (ii) the effect of promoting thesynthesis of ATP, NADPH, a starch, and a protein, and (iii) the effectof promoting carbon fixation. Accordingly, the present invention alsoincludes a method of promoting the growth of a higher plant, a method ofpromoting the synthesis of at least one selected from the groupconsisting of ATP, NADPH, a starch and a protein of a higher plant, anda method of promoting the carbon fixation of a higher plant.

Specifically, these methods are characterized in that they comprise:introducing into the genome of a higher plant a gene encoding a fusedprotein formed by adding a signal peptide consisting of 50 to 80 aminoacid residues to a cytochrome c₆ protein, so as to allow it to expresstherein; and allowing cytochrome c₆ to exist in the thylakoid space ofchloroplast. In these methods, with respect to a fused protein, a geneencoding the above protein, a method of introducing the above gene intoa higher plant genome, various effects obtained from the obtainedtransgenic higher plant, and the like, the aforementioned descriptions,examples, and others, which are given regarding the method of producinga higher plant of the present invention, can also be applied.

The present invention will be more specifically described in thefollowing examples. However, these examples are not intended to limitthe scope of the present invention.

Example 1 Construction of Cytochrome C6 Gene to be Introduced andExpression Vector used for Plants

(1) Preparation of Porphyra yezoensis-Derived Cytochrome c₆ MatureProtein Region Gene

In the present example, a P. yezoensis-derived cytochrome c₆ gene wasacquired by cloning, and it was then ligated to a vector, so as toprepare a plasmid. Using this plasmid as a template, a P.yezoensis-derived cytochrome c₆ mature protein region gene that was DNAof interest in this example was amplified. Thereafter, electrophoresiswas carried out. A gene of interest was extracted by gel extraction, andit was then digested with restriction enzymes (SacI and PstI), so as toprepare a gene.

The reaction solution with the following composition was prepared in a0.5-ml assist PCR tube.

Template (plasmid DNA (overall   1.0 μl length of cytochrome c₆))Primer (1) (10 pmol/μl)  1.0 μl Primer (2) (10 pmol/μl)  1.0 μl 10 ×Ex Taq buffer (Mg²⁺ plus)  2.5 μl (Mg²⁺ concentration: 20 mM)dNTP Mixture (2.5 mM each)  2.0 μl DMSO 1.25 μl Distilled water 16.1 μlTaKaRa Ex Taq (5 U/μl) 0.15 μl Total volume 25.0 μl Primer (1):PYC6-C-term SacI (10 pmol/μl) (30 mer, GC-Cont.: 43.3%) 5′-GGA GCT CTTACC AAC CTT TTT CAG ATT GAG-3′ (SEQ ID NO: 7) Primer (2): Cyt-SD (10pmol/μl) (29 mer, GC-Cont.: 48.2%) 5′-CCG CGG AGA CGT TAA ATT GAA GAAGAA GC-3′ (SEQ ID NO: 8)

15 μl of mineral oil was laminated on the above reaction solution.Thereafter, a reaction was carried out using PTC-100™ ProgrammableThermal Controller. A reaction consisting of 94° C.-48 seconds, 62°C.-48 seconds, and 72° C.-1 minute 24 seconds was defined as 1 cycle. 31cycles of reactions were carried out.

After completion of the reaction, confirmation was carried out by 2%(w/v) agarose gel electrophoresis. 4 μl of a gel loading buffer wasadded to 20 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TBE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera.

A band that was considered to be DNA of interest was cut out of 2% (w/v)agarose gel, and it was then minced as finely as possible. The gel wasplaced in Amicon Ultra free (registered trade mark) DA, and it was thencentrifuged at 7,300 rpm at 4° C. for 10 minutes. After completion ofthe centrifugation, Ultra free MC (a column portion on the upper side)was removed, and to the eluted solution, an equal amount of TE-saturatedPhenol was added. Thereafter, the mixture was fully stirred using avortex, followed by centrifugation at 14,000 rpm at 4° C. for 10minutes. The water layer was transferred into a new 1.5 mlmicrocentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of99.5% ethanol were then added thereto. After the mixture had been fullystirred, it was left at −20° C. for 30 minutes. Thereafter, theresultant was centrifuged at 14,000 rpm at 4° C. for 10 minutes, and thesupernatant was then discarded. 500 μl of 75% ethanol was added to theprecipitate, and the precipitate was then fully washed. The resultantwas further centrifuged at 14,000 rpm at 4° C. for 5 minutes, and thesupernatant was then discarded. The precipitate was air-dried to acertain extent, and it was then dissolved in 50 μl of TE.

Subsequently, a sample having the following composition was prepared ina 1.5 ml microcentrifuge tube. The sample was then incubated at 37° C.for 120 minutes, so that it was digested with the restriction enzymeSacI.

Sample obtained after gel extraction  5.0 μl 10 × L Buffer  2.0 μl SacI 0.5 μl Distilled water 12.5 μl Total volume 20.0 μl

To a sample solution obtained after incubation, an equal amount ofTE-saturated Phenol was added. Thereafter, the mixture was fully stirredusing a vortex, followed by centrifugation at 14,000 rpm at 4° C. for 10minutes. The water layer was transferred into a new 1.5 mlmicrocentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of99.5% ethanol were then added thereto. After the mixture had been fullystirred, it was left at −20° C. for 30 minutes. Thereafter, theresultant was centrifuged at 14,000 rpm at 4° C. for 10 minutes, and thesupernatant was then discarded. 500 μl of 75% ethanol was added to theprecipitate, and the precipitate was then fully washed. The resultantwas further centrifuged at 14,000 rpm at 4° C. for 5 minutes, and thesupernatant was then discarded. The precipitate was air-dried to acertain extent, and it was then dissolved in 20 μl of TE.

Subsequently, a sample having the following composition was prepared ina 1.5 ml microcentrifuge tube. The sample was then incubated at 37° C.for 120 minutes, so that it was digested with the restriction enzymePstI.

Sample after digestion with restriction enzyme 5.0 μl 10 × H Buffer 2.0μl PstI 0.5 μl Distilled water 12.5 μl  Total volume 20.0 μl 

After completion of the reaction, confirmation was carried out by 2%(w/v) agarose gel electrophoresis. 4 μl of a gel loading buffer wasadded to 20 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TBE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera. Thereafter, a band that was considered to be DNAof interest was cut out of 2% (w/v) agarose gel, and it was then mincedas finely as possible. The gel was placed in Amicon Ultra free(registered trade mark) DA, and it was then centrifuged at 7,300 rpm at4° C. for 10 minutes. After completion of the centrifugation, Ultra freeMC (a column portion on the upper side) was removed, and to the elutedsolution, an equal amount of TE-saturated Phenol was added. Thereafter,the mixture was fully stirred using a vortex, followed by centrifugationat 14,000 rpm at 4° C. for 10 minutes. The water layer was transferredinto a new 1.5 ml microcentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH5.2) and 2 vol. of 99.5% ethanol were then added thereto. After themixture had been fully stirred, it was left at −20° C. for 30 minutes.Thereafter, the resultant was centrifuged at 14,000 rpm at 4° C. for 10minutes, and the supernatant was then discarded. 500 μl of 75% ethanolwas added to the precipitate, and the precipitate was then fully washed.The resultant was further centrifuged at 14,000 rpm at 4° C. for 5minutes, and the supernatant was then discarded. The precipitate wasair-dried to a certain extent, and it was then dissolved in 20 μl of TE.

The following results were obtained.

Using a plasmid, into which a P. yezoensis-derived cytochrome c₆ gene(overall length) had been inserted, as a template, a cytochrome c₆mature protein region (PYC6) gene was amplified by PCR (FIG. 4).Thereafter, the PCR product was subjected to 2% (w/v) agarose gel (TBE)electrophoresis. As a result, a single band could be confirmed around357 by (FIG. 5). This band was considered to be a PYC6 gene. Thus, thisPYC6 gene was digested with two restriction enzymes (SacI and PstI), andthe resultant was then subjected to 2% (w/v) agarose gel (TBE)electrophoresis. A band around 265 by that was considered to be the PYC6gene (having Sad and PstI sites) was extracted from the gel, and it wasthen subjected to phenol extraction and ethanol precipitation, so as toobtain a PYC6 gene having Sad and PstI sites.

(2) Addition of Peptide Gene Having Ability to Pass Through ChloroplastEnvelope and Thylakoid Membrane to Cytochrome c₆

In this section, a peptide gene having ability to pass through achloroplast envelope and a thylakoid membrane, which had been subjectedto a treatment with the restriction enzymes BamHI and PstI anddephosphorylation (BAP treatment), was ligated to a P. yezoensis-derivedcytochrome c₆ mature protein region gene, which had been treated withthe restriction enzymes Sad and PstI. Thereafter, the ligated productwas further ligated to a cloning vector (pBluescript (registered trademark) II SK+), followed by subcloning. Thereafter, the nucleotidesequence of a cytochrome c₆ (sp+PYC6) to be introduced into a plant wasconfirmed. The following universal primers were used in DNA sequencing.

Universal FITC Forward primer (M13 Fw primer) (2 pmol/μl) (SEQ ID NO: 9)5′-CGC CAG GGT TTT CCC AGT CAC GAC-3′Universal FITC Reverse primer (M13 Rv primer) (2 pmol/μl)(SEQ ID NO: 10) 5′-GAG CGG ATA ACA ATT TCA CAC AGG-3′(2-1) Preparation of Cytochrome c₆ Gene to be Introduced into Plant

A reaction solution with the following composition was prepared.

Peptide gene having ability to pass through chloroplast 4.0 μl envelopeand thylakoid membrane (BAP-treated) P. yezoensis cytochrome c₆ matureprotein region gene 4.0 μl 10 × Ligation Buffer 2.0 μl 2 mg/ml BSASolution 2.5 μl Enzyme Solution (T₄ DNA Ligase) 1.0 μl Distilled water6.5 μl Total volume 20.0 μl 

The prepared reaction solution was incubated in a low temperatureincubator (16° C.) overnight for a ligation reaction. Subsequently,alkaline phosphatase was added to the solution, so as to carry outdephosphorylation. A sample having the following composition wasprepared in a 1.5 ml microcentrifuge tube, and it was then incubated at37° C. for 3 hours, so as to carry out dephosphorylation using BacterialAlkaline Phosphatase.

Sample obtained after ligation 10.0 μl 10 × BAP Buffer 10.0 μl BacterialAlkaline Phosphatase  2.5 μl (BAP) Distilled water 77.5 μl Total volume100.0 μl 

To the sample solution obtained after incubation, an equal amount ofTE-saturated Phenol was added. Thereafter, the mixture was fully stirredusing a vortex, followed by centrifugation at 14,000 rpm at 4° C. for 10minutes. The water layer was transferred into a new 1.5 mlmicrocentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of99.5% ethanol were then added thereto. After the mixture had been fullystirred, it was left at −20° C. for 30 minutes. Thereafter, theresultant was centrifuged at 14,000 rpm at 4° C. for 10 minutes, and thesupernatant was then discarded. 500 μl of 75% ethanol was added to theprecipitate, and the precipitate was then fully washed. The resultantwas further centrifuged at 14,000 rpm at 4° C. for 5 minutes, and thesupernatant was then discarded. The precipitate was air-dried to acertain extent, and it was then dissolved in 20 μl of TE.

(2-2) Preparation of Cloning Vector pBluescript (Registered Trade Mark)II SK+

A sample having the following composition was prepared in a 1.5 mlmicrocentrifuge tube, and it was then incubated at 37° C. for 5 hours,so that it was digested with the restriction enzyme SacI.

pBluescript (registered trade mark) II SK+ 20 μl 10 × L Buffer  5 μlSacI  2 μl Distilled water 23 μl Total volume 50 μl

To the sample solution obtained after incubation, an equal amount ofTE-saturated Phenol was added. Thereafter, the mixture was fully stirredusing a vortex, followed by centrifugation at 14,000 rpm at 4° C. for 10minutes. The water layer was transferred into a new 1.5 mlmicrocentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of99.5% ethanol were then added thereto. After the mixture had been fullystirred, it was left at −20° C. for 30 minutes. Thereafter, theresultant was centrifuged at 14,000 rpm at 4° C. for 10 minutes, and thesupernatant was then discarded. 500 μl of 75% ethanol was added to theprecipitate, and the precipitate was then fully washed. The resultantwas further centrifuged at 14,000 rpm at 4° C. for 5 minutes, and thesupernatant was then discarded. The precipitate was air-dried to acertain extent, and it was then dissolved in 20 μl of TE.

Subsequently, a sample having the following composition was prepared ina 1.5 ml microcentrifuge tube, and it was then incubated at 37° C. for 5hours, so that it was digested with the restriction enzyme BamHI.

Sample obtained after digestion with restriction enzyme 20 μl 10 × HBuffer  5 μl BamHI  2 μl Distilled water 23 μl Total volume 50 μl

After completion of the reaction, confirmation was carried out by 1.5%(w/v) agarose gel electrophoresis. 4 μl of a gel loading buffer wasadded to 20 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TAE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera. Thereafter, a band that was considered to be DNAof interest was cut out of 1.5% (w/v) agarose gel, and 100 μg of the gelwas converted to 100 μA 3 vol. of NaI solution was added to the gel, andthe mixture was then fully stirred. Thereafter, the mixture wasincubated at 55° C. for 5 minutes, so as to dissolve the gel. 10 μl ofGLASSMILK was added to the resultant, and the mixture was intensivelystirred at room temperature for 15 minutes, followed by centrifugationat 14,000 rpm at 4° C. for 5 seconds, so as to recover a precipitate.300 μl of NEW WASH was added to the precipitate, and the mixture wasthen fully stirred. The resultant was centrifuged at 14,000 rpm at 4° C.for 5 seconds, so as to recover a precipitate. This operation wasrepeated 3 times, and the obtained precipitate was then dried. The driedprecipitate was dissolved in 20 μl of distilled water. Thereafter, theobtained solution was transferred into a 0.5-ml assist PCR tube, and itwas then centrifuged at 14,000 rpm at 4° C. for 30 seconds. The obtainedsupernatant was transferred into a new 1.5 ml microcentrifuge tube, and1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of 99.5% ethanol were thenadded thereto. After the mixture had been fully stirred, it was left at−20° C. for 30 minutes. Thereafter, the resultant was centrifuged at14,000 rpm at 4° C. for 10 minutes, and the supernatant was thendiscarded. 500 μl of 75% ethanol was added to the precipitate, and theprecipitate was then fully washed. The resultant was further centrifugedat 14,000 rpm at 4° C. for 5 minutes, and the supernatant was thendiscarded. The precipitate was air-dried to a certain extent, and it wasthen dissolved in 20 μl of TE.

(2-3) Subcloning of Cytochrome c₆ Gene to be Introduced into Plants

The cytochrome c₆ gene was subcloned according to a known method(Hanahan, D., J. Mol. Biol., 166(4), 557-580 (1983); and Hiroki Nakayamaet al., Bio Illustrated II, Idenshi Kaiseki no Kiso (Basic GeneAnalyses), Shujunsha Co., Ltd., 83-88 (1996)).

The gene to be introduced into plants and a cloning vector were preparedsuch that they had the following compositions.

Cytochrome c₆ to be introduced (after it was 5 μl digested with BamHIand SacI) (BAP-treated) pBluescript (registered trade mark) II SK+ 5 μl(digested with BamHI and SacI) 10 × Ligation Buffer 2 μl 2 mg/ml BSASolution 2.5 μl   Enzyme Solution (T₄ DNA Ligase) 1 μl Distilled water4.5 μl   Total volume 20 μl 

The prepared reaction solution was incubated in a low temperatureincubator (16° C.) overnight for a ligation reaction. Escherichia coliwas transformed as follows. First, 150 μl of competent cells (DH5α) weremelted on ice to a certain extent, and 4 μl of the ligated reactionsolution was then gently added thereto. The mixture was softly stirredwith the tip of a chip, and it was then left on ice for 30 minutes.After it had left for 30 minutes, it was heat shocked at 42° C. for 20seconds. Thereafter, the resultant was left at rest on ice for 3minutes. Thereafter, each of 100 μl of and 50 μl of cell mass solutions(Vector:Insert=1:1, and 1:3) was applied on the entire surface of anLB-Ampicillin-X-Gal-IPTG plate, using a cell inoculation loop, and itwas then cultured at 37° C. overnight.

(2-4) Preparation of Plasmid by Alkaline SDS Method

The alkaline SDS method was carried out according to known means (Weiss.B, et al., J. Biol. Chem., 243, 4543-4555 (1968); and Birnboim, H. C.,Methods Enzymol., 100, 243 (1983)). That is to say, a cell mass solutionwas subjected to a shaking culture in 3 ml of LB-Amplicillin liquidmedium at 200 rpm at 37° C. for 16 hours, and it (1.5 ml each) was thendispensed into a 1.5 ml microcentrifuge tube, followed by centrifugationat 6,000 rpm at 4° C. for 10 minutes. Thereafter, the supernatant waseliminated by aspiration with an aspirator. 100 μl of Solution I (50 mMglucose-25 mM Tris-HCl (pH 8.0)-10 mM EDTA) was added to the precipitate(cell mass), and the obtained mixture was fully stirred with a vortex.Thereafter, 200 μl of Solution II was added thereto, and the obtainedsolution was stirred by gently turning it over. The solution was left atroom temperature for 5 minutes, and 150 μl of Solution III (0.2 NNaOH-1% SDS) was further added thereto. The obtained mixture was left onice for 30 minutes, and 100 μl of chloroform was then added thereto,followed by centrifugation at 14,000 rpm at 4° C. for 10 minutes.Thereafter, the supernatant was recovered in a new tube, and a doublevolume of 99.5% ethanol was then added thereto. The obtained mixture wasstirred by gently turning it over, and it was then left at roomtemperature for 15 minutes. Thereafter, 500 μl of 70% ethanol was addedto the reaction solution, and the obtained mixture was then centrifugedat 14,000 rpm at 4° C. for 10 minutes. The obtained precipitate wasdried with TOMY MICRO Vac for 3 to 5 minutes, and it was then dissolvedin 20 μl of TE.

In order to confirm that DNA of interest had been incorporated into theobtained plasmid, a sample having the following composition was preparedin a 1.5 ml microcentrifuge tube. It was then incubated at 37° C. for120 minutes, so that it was digested with the restriction enzymes Sadand BamHI.

Plasmid DNA 5.0 μl 10 × L Buffer 2.0 μl SacI 0.5 μl BamHI 0.5 μl 1 mg/mlRNase A 0.5 μl Distilled water 11.5 μl  Total volume 20.0 μl 

After completion of the reaction, confirmation was carried out by 2%(w/v) agarose gel electrophoresis. 2 μl of a gel loading buffer wasadded to 10 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TBE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera. As a result, with respect to only the sample intowhich the DNA of interest had been incorporated, 100 μl out of theremaining cell mass solution (1.5 ml) obtained after completion of theculture in 3 ml of LB-Ampicillin liquid medium was inoculated into 5 mlof LB-Ampicillin liquid medium, followed by a shaking culture at 200rpm, at 37° C. for 16 hours.

(2-5) Preparation of Plasmid with QIAGEN Spin Miniprep Kit

A cell mass solution obtained by the culture in 5 ml of LB-Ampicillinliquid medium was centrifuged at 6,000 rpm at 4° C. for 10 minutes, andthe supernatant was then eliminated by aspiration with an aspirator. 250μl of Buffer P1 included with the kit (QIAGEN Spin Miniprep Kit(250)(QIAGEN)) was added to the precipitate (cell mass), and it was fullysuspended therein.

It is to be noted that the QIAGEN Spin Miniprep Kit(250) (QIAGEN)comprises a Collection tube, a QIA prep Spin Column, Buffer P1, BufferP2, Buffer N3, Buffer PB, Buffer PE, and Buffer EB.

250 μl of Buffer P2 was added to the above suspension, and the obtainedmixture was stirred by gently turning it over, until the solution becamehomogeneous. Thereafter, 350 μl of Buffer N3 was further added to thesolution, and the obtained mixture was stirred by gently turning itover, followed by centrifugation at 13,000 rpm at 4° C. for 10 minutes.The obtained supernatant was transferred into a QIA prep spin column,which had previously been set in a Collection tube, and it was thencentrifuged at 13,000 rpm at 4° C. for 1 minute. The filtrateaccumulated in the Collection tube was discarded, and 500 μl of BufferPB was then added to the QIA prep spin column, followed bycentrifugation at 13,000 rpm at 4° C. for 1 minute. Thereafter, thefiltrate was discarded again. 750 μl of Buffer PE was further added tothe QIA prep spin column, followed by centrifugation at 13,000 rpm at 4°C. for 1 minute. Thereafter, the filtrate was discarded. Centrifugationwas again carried out at 13,000 rpm at 4° C. for 1 minute, and only theQIA prep spin column was transferred to a new 1.5 ml microcentrifugetube. 50 μl of Buffer EB was added to the center of the QIA prep spincolumn, and it was then left at room temperature for 1 minute, followedby centrifugation at 13,000 rpm at 4° C. for 1 minute. The obtainedfiltrate was defined as a plasmid sample.

In order to confirm that DNA of interest had been incorporated into theobtained plasmid, a sample having the following composition was preparedin a 1.5 ml microcentrifuge tube, and it was then incubated at 37° C.for 120 minutes, so that it was digested with the restriction enzymesSad and BamHI.

Plasmid DNA 5.0 μl 10 × L Buffer 2.0 μl SacI 0.5 μl BamHI 0.5 μlDistilled water 12.0 μl  Total volume 20.0 μl 

After completion of the reaction, confirmation was carried out by 2%(w/v) agarose gel electrophoresis. 2 μl of a gel loading buffer wasadded to 10 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TBE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera. Herein, a sample, into which the DNA of interesthad been incorporated and wherein unity had been confirmed, was definedas a sequence sample.

(2-6) Determination of Nucleotide Sequence according to Dideoxy Method

Nucleotide sequences were determined by a known method using anautosequencer (Sanger, F., Sanger, F., Determination of nucleotidesequence in DNA., Science, 214, 1205-1210 (1981)). After determinationof such nucleotide sequences, data were analyzed using analysissoftware, GENETYX-MAC.

(2-7) Results

An sp gene, which had been subjected to digestion with two restrictionenzymes (BamHI and PstI) and dephosphorylation (BAP treatment), wasligated to a PYC6 gene, which had been digested with two restrictionenzymes (SacI and PstI). The ligated product was treated with BAP again,and the resultant was then subjected to phenol extraction and ethanolprecipitation, so as to produce a cytochrome c₆(sp+PYC6) gene to beintroduced into plants.

In order to determine the nucleotide sequence of this sp+PYC6 gene, thegene was ligated to a cloning vector (pBluescript II SK+ (FIG. 6)),which had been digested with two restriction enzymes (BamHI and SacI),followed by subcloning. Thereafter, the resultant was sequenced by thedideoxy method, so as to determine the entire nucleotide sequence (474bp) of the sp+PYC6 gene (FIG. 7).

The sequence results of the sp+PYC6 gene were compared with those of theknown sp gene or PYC6 gene. As a result, it was revealed that thesequence of the sp+PYC6 gene was completely identical to that of theknown sp gene or PYC6 gene, with the exception that T (thymine) atposition 103 in the signal peptide sequence had been mutated to A(adenine). In addition, restriction enzyme sites added into primersequences were also confirmed (FIG. 7). Due to the thus confirmednucleotide mutation, the encoded amino acid Ser was converted to Thr.However, their properties and molecular weights were similar, and suchconversion had no influence upon the secondary structure (α-helix,etc.). Thus, since such conversion has no influence upon the function asa signal peptide, such as membrane transfer, this sample (sp+PYC6) wasused in the subsequent experiment.

(3) Production of Expression Vector for Cytochrome c₆ to be Introducedinto Plants

In the present example, a cytochrome c₆(sp+PYC6) gene to be introducedinto plants, whose nucleotide sequence had been confirmed, was insertedinto an expression vector pBI121, so as to construct an expressionvector (pBI cyt c₆) used for cytochrome c₆ to be introduced into plants.

(3-1) Preparation of Plant Expression Vector pBI121

A sample having the following composition was prepared in 1.5 mlmicrocentrifuge tube, and it was then incubated at 37° C. for 5 hours,so that it was digested with the restriction enzyme SacI.

pBI121 20 μl 10 × L Buffer  5 μl SacI  2 μl Distilled water 23 μl Totalvolume 50 μl

To the sample solution obtained after the incubation, an equal amount ofTE-saturated Phenol was added. Thereafter, the mixture was fully stirredusing a vortex, followed by centrifugation at 14,000 rpm at 4° C. for 10minutes. The water layer was transferred into a new 1.5 mlmicrocentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of99.5% ethanol were then added thereto. After the mixture had been fullystirred, it was left at −20° C. for 30 minutes. Thereafter, theresultant was centrifuged at 14,000 rpm at 4° C. for 10 minutes, and thesupernatant was then discarded. 500 μl of 75% ethanol was added to theprecipitate, and the precipitate was then fully washed. The resultantwas further centrifuged at 14,000 rpm at 4° C. for 5 minutes, and thesupernatant was then discarded. The precipitate was air-dried to acertain extent, and it was then dissolved in 20 μl of TE.

Subsequently, a sample having the following composition was prepared ina 1.5 ml microcentrifuge tube. The sample was then incubated at 37° C.for 5 minutes, so that it was digested with the restriction enzymeBamHI.

Sample obtained after digestion with 20 μl restriction enzyme 10 × HBuffer  5 μl BamHI  2 μl Distilled water 23 μl Total volume 50 μl

After completion of the reaction, confirmation was carried out by 1.5%(w/v) agarose gel electrophoresis. 4 μl of a gel loading buffer wasadded to 20 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TAE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera. Thereafter, a band that was considered to be DNAof interest was cut out of 1.5% (w/v) agarose gel, and 100 μg of the gelwas converted to 100 μA 3 vol. of NaI solution was added to the gel, andthe mixture was then fully stirred. Thereafter, the mixture wasincubated at 55° C. for 5 minutes, so as to dissolve the gel. 10 μl ofGLASSMILK was added to the resultant, and the mixture was intensivelystirred at room temperature for 15 minutes, followed by centrifugationat 14,000 rpm at 4° C. for 5 seconds, so as to recover a precipitate.300 μl of NEW WASH was added to the precipitate, and the mixture wasthen fully stirred. The resultant was centrifuged at 14,000 rpm at 4° C.for 5 seconds, so as to recover a precipitate. This operation wasrepeated 3 times, and the obtained precipitate was then dried. The driedprecipitate was dissolved in 20 μl of distilled water. Thereafter, theobtained solution was transferred into a 0.5-ml assist PCR tube, and itwas then centrifuged at 14,000 rpm at 4° C. for 30 seconds. The obtainedsupernatant was transferred into a new 1.5 ml microcentrifuge tube, and1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of 99.5% ethanol were thenadded thereto. After the mixture had been fully stirred, it was left at−20° C. for 30 minutes. Thereafter, the resultant was centrifuged at14,000 rpm at 4° C. for 10 minutes, and the supernatant was thendiscarded. 500 μl of 75% ethanol was added to the precipitate, and theprecipitate was then fully washed. The resultant was further centrifugedat 14,000 rpm at 4° C. for 5 minutes, and the supernatant was thendiscarded. The precipitate was air-dried to a certain extent, and it wasthen dissolved in 20 μl of TE.

(3-2) Preparation of Cytochrome c₆ Expression Vector (Pbi Cyt c₆) Usedfor Expression in Plants

The cytochrome c₆(sp+PYC6) gene to be introduced, which had beenproduced above, was used to prepare a reaction solution having thefollowing composition.

sp+PYC6 gene (after digestion with BamHI and SacI) 4.0 μl (BAP-treated)pBI121 (after digestion with BamHI and SacI) 8.0 μl 10 × Ligation Buffer2.0 μl 2 mg/ml BSA Solution 2.5 μl Enzyme Solution (T₄ DNA Ligase) 1.0μl Distilled water 2.5 μl Total volume 20.0 μl 

The prepared reaction solution was incubated in a low temperatureincubator (16° C.) overnight for a ligation reaction.

The results are shown below.

The sp+PYC6 gene (which had been digested with BamHI and Sad and hadbeen treated with BAP), whose nucleotide sequence had been confirmed,was ligated to a plant expression vector (pBI121), which had beendigested with two restriction enzymes (BamHI and SacI), so as to preparea cytochrome c₆ expression vector (pBI cyt c₆) to be introduced intoplants.

Example 2 Production of Transgenic Plant Cytochrome c6-IntroducedArabidopsis thaliana

(1) Preparation of Agrobacterium tumefaciens used in Plant Infection(1-1) Preparation of Cytochrome c₆ exprzession Vector (pBI cytc₆)-Introduced Escherichia coli

In the present example, Escherichia coli HB101 used as a host wastransformed with a plant expression vector (pBI cyt c₆), into which acytochrome c₆ gene to be introduced into plants had been inserted.Thereafter, several colonies were selected, and they were then culturedin 3 ml of LB-Kanamycin liquid medium. A plasmid was prepared by thealkaline SDS method, so as to confirm construction of the vector and thepresence or absence of transformation of the host Escherichia coliHB101. Escherichia coli was transformed as follows. First, 150 μl ofcompetent cells (HB101) were melted on ice to a certain extent, and 4 μlof the ligated reaction solution was then gently added thereto. Themixture was softly stirred with the tip of a chip, and it was then lefton ice for 30 minutes. After it had left for 30 minutes, it was heatshocked at 42° C. for 20 seconds. Thereafter, the resultant was left atrest on ice for 3 minutes. Thereafter, each of 100 μl of and 50 μl ofcell mass solutions was applied on the entire surface of an LB-Kanamycinplate, using a cell inoculation loop, and it was then cultured at 37° C.overnight.

After completion of the culture, 20 clones were randomly picked up fromtransformants (colonies), and the thus obtained colonies were theninoculated into 3 ml of LB-Kanamycin medium, and they were thensubjected to a shaking culture at 200 rpm at 37° C. for 16 hours.Thereafter, the cultured cell mass solution (1.5 ml each) was thendispensed into a 1.5 ml microcentrifuge tube, followed by centrifugationat 6,000 rpm at 4° C. for 10 minutes. Thereafter, the supernatant waseliminated by aspiration with an aspirator. 100 μl of Solution I (theabove-mentioned) was added to the precipitate (cell mass), and theobtained mixture was fully stirred with a vortex. Thereafter, 200 μl ofSolution II (0.2 N NaOH-1% SDS) was added thereto, and the obtainedsolution was stirred by gently turning it over. The solution was left atroom temperature for 5 minutes, and 150 μl of Solution III (theabove-mentioned) was further added thereto. The obtained mixture wasleft on ice for 30 minutes, and 100 μl of chloroform was then addedthereto, followed by centrifugation at 14,000 rpm at 4° C. for 10minutes. Thereafter, the supernatant was recovered in a new tube, and adouble volume of 99.5% ethanol was then added thereto. The obtainedmixture was stirred by gently turning it over, and it was then left atroom temperature for 15 minutes. Thereafter, 500 μl of 70% ethanol wasadded to the reaction solution, and the obtained mixture was thencentrifuged at 14,000 rpm at 4° C. for 10 minutes. The obtainedprecipitate was dried with TOMY MICRO Vac for 3 to 5 minutes, and it wasthen dissolved in 20 μl of TE.

In order to confirm that DNA of interest had been incorporated into theobtained plasmid, a sample having the following composition was preparedin a 1.5 ml microcentrifuge tube, and it was then incubated at 37° C.for 120 minutes, so that it was digested with the restriction enzymesBamHI and SacI.

Plasmid DNA 5.0 μl 10 × L Buffer 2.0 μl BamHI 0.5 μl SacI 0.5 μl 1 mg/mlRNase A 0.5 μl Distilled water 11.5 μl  Total volume 20.0 μl 

After completion of the reaction, confirmation was carried out by 1.5%(w/v) agarose gel electrophoresis. 2 μl of a gel loading buffer wasadded to 10 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TAE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera.

The results are shown below. Escherichia coli HB101 used as a host wastransformed with a cytochrome c₆ expression vector (pBI cyt c₆) used forexpression in plants (FIG. 8). Thereafter, 20 clones were randomlypicked up from transformants (colonies). As a result, in 3 out of the 20clones, a single band could be confirmed around the size (474 bp) thatwas considered to be an sp+PYC6 gene. Thus, it was revealed that thesp+PYC6 gene had been inserted into this plasmid. The Escherichia coliHB101 having this plasmid was used in the subsequent experiment.

(1-2) Preparation and Selection of Transgenic Agrobacterium tumefaciens

In this section, triparental mating was carried out to prepareAgrobacterium tumefaciens used as a host that was necessary forintroduction of a gene of interest into a higher plant A. thaliana.Thereafter, several colonies were selected from the obtainedtransformants, and they were then cultured in 3 ml of LB-Kanamycinliquid medium, followed by preparation of a plasmid by the alkaline SDSmethod, so as to confirm the presence or absence of transformation ofthe host Agrobacterium.

(1-2-1) Culture of Various Types of Strains

(a) Agrobacterium tumefaciens (LBA4404)*pAL4404 Maintenance

A glycerol stock solution of the above strain was applied in a linedrawing manner on the entire surface of a YEP-Streptomycin agar medium(plate) (30 μg/ml Streptomycin), using a platinum loop. It was thencultured at 30° C. for approximately 40 hours. After completion of theculture, a colony was picked up, and it was then subjected to a shakingculture in 5 ml of YEP-Streptomycin (30 μg/ml Streptomycin) medium at200 rpm at 30° C. for 30 hours.

(b) Escherichia coli (HB101)*pRK2013 Maintenance

A glycerol stock solution of the above strain was applied in a linedrawing manner on the entire surface of an LB-Kanamycin agar medium(plate) (40 μg/ml Kanamycin), using a platinum loop. It was thencultured at 37° C. for 16 hours. After completion of the culture, acolony was picked up, and it was then subjected to a shaking culture in5 ml of LB-Kanamycin (40 μg/ml Kanamycin) medium at 200 rpm at 37° C.for 16 hours.

(c) Escherichia coli (HB101)*pBI cyt c₆ Maintenance

A glycerol stock solution of the above strain was applied in a linedrawing manner on the entire surface of an LB-Kanamycin agar medium(plate) (40 μg/ml Kanamycin), using a platinum loop. It was thencultured at 37° C. for 16 hours. After completion of the culture, acolony was picked up, and it was then subjected to a shaking culture in5 ml of LB-Kanamycin (40 μg/ml Kanamycin) medium at 200 rpm at 37° C.for 16 hours.

(1-2-2) Triparental Mating

Triparental mating was carried out according to a known method (AtsushiKomamine & Koji Nomura, “Seibutsu Kagaku Jikken Ho 41 (BiochemicalExperimental methods 41),” Syokubutsu Saibo Kogaku Nyumon (Introductionof Plant Cell Technology), Japan Scientific Societies Press, 298-302(1998)). That is, each of 3 types of strain solutions (strains having A.tumefaciens (LBA4404)*pAL4404, E. coli (HB101)*pBI cyt c₆, and E. coli(HB101)*pRK2013), which had been subjected to a shaking culture in 5 mlof YEP-Sm. liquid medium or LB-Km. liquid medium, was centrifuged at5,000 rpm at 4° C. for 5 minutes. Thereafter, the supernatant waseliminated by aspiration with an aspirator. For washing, the liquidmedium (YEP medium or LB medium) that had been used in the culture wasadded to the precipitate (cell mass), and the obtained mixture was fullystirred. Thereafter, the resultant was centrifuged again at 5,000 rpm at4° C. for 5 minutes. After completion of the centrifugation, the cellmass was dissolved in 3 ml of the liquid medium used in the culture (YEPmedium or LB medium). Subsequently, 30 μl of each cell mass solution wasdropped on a Min A-Kanamycin agar medium (plate) such that it waslaminated thereon. It was then left at rest, so that it was cultured at30° C. for 3 days. After completion of the culture, a cell mass growingfrom the Min A-Kanamycin agar medium (plate) as a minimal medium was allscraped off using a platinum loop, and the thus obtained cell mass wasthen dissolved in 200 μl of 10 mM MgSO₄. Thereafter, the cell masssolution was applied in a line drawing manner on the entire surface of aMin A-Kanamycin agar medium (plate) again using a platinum loop, and itwas then cultured at 30° C. for 3 days. After completion of the culture,a colony was picked up, and it was then subjected to a shaking culturein 5 ml of YEP-Kanamycin (40 μg/ml Kanamycin) medium at 200 rpm at 30°C. for 40 hours.

(1-2-3) Preparation of Ti Plasmid by Alkaline SDS Method

The cell mass solution (1.5 ml each), which had been subjected to ashaking culture in 5 ml of YEP-Kanamycin (40 μg/ml Kanamycin) liquidmedium at 200 rpm at 30° C. for 40 hours, was dispensed in a 1.5 mlmicrocentrifuge tube. Thereafter, the above cell mass solution wascentrifuged at 5,000 rpm at 4° C. for 10 minutes, and the supernatantwas then eliminated by aspiration with an aspirator. Thereafter, 1 ml ofS-buffer was added to the precipitate (cell mass), and the obtainedmixture was fully stirred using a vortex. The mixture was centrifuged at8,000 rpm at 4° C. for 10 minutes, and the supernatant was theneliminated by aspiration with an aspirator.

S-buffer was prepared by weighing 13.512 g of C₆H₁₂O₆ (glucose (M.W.:180.16)), adding 12.5 ml of 1 M Tris-HCl (pH 8.0) and 10 ml of 0.5M EDTA(pH 8.0) thereto, and adjusting the volume to 500 ml with extra purewater, followed by an autoclave treatment (121° C., 10 minutes).

100 μl of S-buffer and 5 μl of 10 mg/ml lysozyme were added to theprecipitate again, and the obtained mixture was left at room temperaturefor 10 minutes. Thereafter, 200 μl of Solution II and 30 μl ofTE-saturated Phenol were added thereto, and the obtained solution wasstirred by gently turning it over. The solution was left at roomtemperature for 5 minutes, and 150 μl of Solution III was further addedthereto. The obtained mixture was left at −20° C. for 15 minutes, andthe resultant was then centrifuged at 12,000 rpm at 4° C. for 10minutes. The water layer was transferred into a new 1.5 mlmicrocentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of99.5% ethanol were then added thereto. After the mixture had been fullystirred, it was left at −20° C. for 30 minutes. Thereafter, theresultant was centrifuged at 14,000 rpm at 4° C. for 10 minutes, and thesupernatant was then discarded. 500 μl of 75% ethanol was added to theprecipitate, and the precipitate was then fully washed. The resultantwas further centrifuged at 14,000 rpm at 4° C. for 5 minutes, and thesupernatant was then discarded. The precipitate was air-dried to acertain extent, and it was then dissolved in 20 μl of TE.

1 ml of RNase A was added to the obtained plasmid, and the obtainedmixture was then incubated at 37° C. for 90 minutes so as to conduct anRNase treatment. After completion of the reaction, confirmation wascarried out by 1.5% (w/v) agarose gel electrophoresis. 2 μl of a gelloading buffer was added to 10 μl of the obtained PCR product, and theobtained mixture was used as an electrophoretic sample. Electrophoresiswas carried out at 100 V for 30 minutes using an electrophoresis buffer(1×TAE). After completion of the electrophoresis, the gel was stainedwith 0.1 mg/ml ethidium bromide for 7 minutes, and it was thendecolorized with extra pure water for 10 minutes. The decolorized gelwas placed on a UV transilluminator, and it was then photographed with anonautomatic hooded instant camera.

The following results were obtained. A. tumefaciens (LBA4404) wascultured in a YEP-Sm. Medium, and E. coli (HB101) was cultured in anLB-Km. medium. Thereafter, pBI cyt c₆ was introduced into A. tumefaciens(LBA4404) by triparental mating, so as to transform A. tumefaciens(LBA4404). Subsequently, 10 clones were randomly picked up from thetransgenic A. tumefaciens strains, into which the Ti plasmid (pBI cytc₆) had been introduced by triparental mating. Such colonies werecultured in 3 ml of YEP-Km. medium. Thereafter, a plasmid was preparedby the alkaline SDS method, and it was then subjected to 1% (w/v)agarose gel electrophoresis. As a result, in 1 clone, a band could beconfirmed at a size (13.5 kbp) that was considered to be pBI cyt c₆.Thus, it was revealed that the Ti plasmid (pBI cyt c₆) had beenintroduced into the transgenic A. tumefaciens.

(2) Production of Transgenic Plant (Cytochrome c₆-Introduced Arabidopsisthaliana)(2-1) Cultivation of Arabidopsis thaliana

Vermiculite was placed up to about 9 parts of a 7.5×7.5 round pot, whichhad well washed with water. Subsequently, 500 ml of a solution wasprepared by 2,000 times diluting a HYPONex stock solution with water,and rock wools, which had been sliced into a thickness of approximately1 cm with a cutter, were placed in the above solution. 3 rock wools,which had been well impregnated with water, were placed on each of the7.5×7.5 round pots. A mesh used for the sink in the kitchen was cut intoa size sufficient to cover the mouth of the pot, and it then well washedwith water. Thereafter, the mouth of the pot was covered with the mesh,and it was then bound with a rubber band. Thereafter, a single seed wasinseminated into each of such pots. After insemination of the seed, alow temperature treatment was carried out thereon at 4° C. for 3 days.After completion of the low temperature treatment, the pot wastransferred into a light irradiation incubator used for the growth ofplants, followed by cultivation under 22° C. for long day conditions.

After germination, water was given approximately once 2 days, andHYPONex that had been 1,000 times diluted was given as a liquidfertilizer approximately once a week. Seedlings were thinned out asappropriate. Approximately 3 weeks later, when plant bodies started toform flower buds, pinching was carried out, and the grown plant bodieswere supported with bamboo skewers (supports). When the seeds havecompletely matured, they were collected. The collected seeds werepreserved in a dry state.

(2-2) Infection of Plant Body with Transgenic Agrobacterium tumefaciens

Subsequently, in order to introduce into a higher plant A. thaliana(col-0), cytochrome c₆ (sp+PYC6 gene) to be introduced into plants, atransgenic plant was produced by a vacuum infiltration method, one ofAgrobacterium methods (Bechtold, N., Pelletier, G., Methods in MolecularBiology, 82 (1998)). Specific procedures are as follows.

(2-2-1) Culture of Agrobacterium tumefaciens

A glycerol stock solution of the above strain was applied in a linedrawing manner on the entire surface of a Min A-Km. agar medium (plate)(400 μg/ml Kanamycin), using a platinum loop. It was then cultured at30° C. for 40 hours. After completion of the culture, a colony waspicked up, and it was subjected to a shaking culture (pre-culture) in 10ml of YEP-Km. (40 μg/ml Kanamycin) medium at 200 rpm at 30° C. for 30hours. Thereafter, the total amount of the culture solution was added to100 ml of new YEP-Km. (40 μg/ml Kanamycin) medium, and a shaking culture(main culture) was then carried out at 200 rpm at 30° C. for 30 hours.

(2-2-2) Cultivation of Plant Body, Arabidopsis thaliana (col-0)

The following cultivation conditions were applied from insemination tocollection of seeds of A. thaliana (col-0) used as a plant to betransformed.

Low temperature treatment: leaving at rest at 4° C. (dark place) for 3days

Plant growth temperature: 22° C.

Light irradiation: long day conditions (100 μm⁻² s⁻¹)

Liquid fertilizer: HYPONeX (1/1,000 times diluted)

In the case of a wild-type strain, it germinated 7 days afterinsemination (including 3 days for the low temperature treatment), andthe plant formed flower buds approximately 20 days after germination.Thereafter, the plant was subjected to a pinching treatment. 5 or 6 dayslater, lateral branches began to extend, and at the same time, theopening of a first flower and fructication started. When green podsbecame light brown and began to split in the longitudinal direction,seeds were collected. Approximately 50 seeds were collected from asingle pod. The collected seeds were preserved in a dry state until thetime of insemination.

(2-2-3) Infection of Plant body

A plant body Arabidopsis thaliana (col-0) used to be infected, wascultivated according to the method described in the section (2-2-2)above, and it was allowed to grown until it began to form flower buds.Thereafter, when foliage began to be derived from the stem, the plantwas pinched, so as to eliminate flowers that had already opened andfructified. In this experiment, preparation of this plant body andpreparation of the cell mass culture solution described in the section(2-2-1) were simultaneously carried out.

First, a Kimtowel was placed on a testing bench on which an infiltrationoperation was to be performed.

Subsequently, an A. tumefaciens culture solution was diluted at amagnification of about ⅔ with a suspension medium used in infiltration,and the diluted solution was then poured into a 1-L beaker. The beakerwas set in an aspiration jar. Thereafter, a plant body of A. thaliana,which had been allowed to absorb a sufficient amount of waterimmediately before inoculation such that it did not aspirate anexcessive amount of culture suspension, was then inoculated into a pot.Thereafter, the pot was immobilized upside-down on the beaker, so as toimmerse the plant body in the culture suspension.

An A. tumefaciens culture solution having OD₆₀₀=1.2 to 1.5 was dilutedat a magnification of about ⅔ with a suspension medium used ininfiltration, so as to obtain OD₆₀₀=0.8, and the thus obtained A.tumefaciens culture solution was used in the vacuum infiltration method.In addition, as a result of examination about the vacuum infiltrationtime, such as 30 seconds, 1 minute, 5 minutes, and 10 minutes, it wasfound that 5 minutes was the most suitable as a time required for vacuuminfiltration. That is, the longer such vacuum infiltration time, thepoor the subsequent growth of the plant body that could be obtained. Inthe case of the treatment for 5 minutes, deterioration of the growth wasnot observed, and the number of flowers and the number of fructifiedpods (seeds) were not significantly different from those of a plant bodythat had been treated for 1 minute.

Subsequently, the plant body that had been immersed in the culturesuspension was placed in the aspiration jar, and the pressure was thenreduced using an aspirator. After completion of the depressurization,the plant body was removed from the aspiration jar, and a redundantculture suspension was wiped with a Kimtowel. The plant body was placedsideways on a plastic tray, and water was added dropwise to the edgethereof. Thereafter, the plastic tray was closed, and it was then leftat 20° C. for 2 or 3 days (during this period, no water was given).Thereafter, the pot was raised, and it was then cultivated, as usual, ina light irradiation incubator used for the growth of plants at 22° C.under long day conditions. Water was given approximately once 2 days,and HYPONex that had been 1,000 times diluted was given as a liquidfertilizer approximately once a week. Seedlings were thinned out asappropriate. Approximately 3 weeks later, when plant bodies started toform flower buds, pinching was carried out, and the grown plant bodieswere supported with bamboo skewers (supports). When the seeds havecompletely matured, they were collected. The collected seeds werepreserved in a dry state.

Example 3 Analysis of Introduction of Cytochrome C6 Gene into TransgenicPlant Body and Expression Thereof

(1) Analysis of Gene Introduced into Genomic DNA by PCR Method

In the present example, genomic DNA was extracted from transgenicArabidopsis thaliana. Using the genomic DNA as a template, PCR wascarried out. Introduction of a gene into the plant body was confirmedbased on the presence or absence of amplification of the introducedgene.

(1-1) Extraction of Genomic DNA from Transformant

Approximately 2 or 3 leaves of transgenic Arabidopsis thaliana wereplaced in a mortar that had previously been cooled, and liquid nitrogenwas rapidly added thereto. Thereafter, the plant body was homogenizeduntil it became a powder state. Thereafter, the powder sample was placedin a 1.5 ml microcentrifuge tube, followed by weighing. 1 ml of asolution obtained by adding 2-mercaptoethanol to Wash buffer to a finalconcentration of 0.5% was added to the weighed sample, and the mixturewas fully blended. The mixture was centrifuged at 12,500 rpm at 4° C.for 10 minutes, and the supernatant was then discarded. 300 μl of asolution obtained by adding 2-mercaptoethanol to Solution I to a finalconcentration of 1% was added thereto, and the obtained mixture wasfully blended using a vortex. Subsequently, 30 μl of 1% NaBH₄ and 150 μlof Solution II were added to the resultant, and the obtained mixture wasstirred using a vortex for several seconds. The resultant solution wasincubated at 50° C. for 10 minutes, and 100 μl of Solution III-A and 120μl of Solution III-B included with ISOPLANT II (NIPPON GENE) were thenadded to the solution. The obtained solution was left on ice for 10minutes.

The resultant was centrifuged at 12,500 rpm at 4° C. for 10 minutes, andthe water phase was recovered. Thereafter, 2 vol. of 99.5% ethanol wasadded thereto, and the mixture was immediately centrifuged at 8,000 rpmat room temperature for 1 hour. Thereafter, the supernatant wasdiscarded. 1 ml of 70% ethanol was further added thereto, and themixture was then centrifuged at 8,000 rpm at room temperature for 1minute. Thereafter, the supernatant was discarded. The precipitate wasair-dried to a certain extent, and it was then dissolved in 50 μl of TE.1 μl of 1 mg/ml RNase A was added to the solution, and the mixture wasthen incubated at 37° C. for 30 minutes. Thereafter, in order to confirmextraction of genomic DNA, 1 μl of a gel loading buffer was added to 5μl of the obtained genomic DNA, and using MindIII as a marker, 1.5%(w/v) agarose gel electrophoresis was carried out. The electrophoresiswas carried out using an electrophoresis buffer (1×TAE) at 100 V for 30minutes. After completion of the electrophoresis, the gel was stainedwith 0.1 mg/ml ethidium bromide for 7 minutes, and it was thendecolorized with extra pure water for 10 minutes. The decolorized gelwas placed on a UV transilluminator, and it was then photographed with anonautomatic hooded instant camera.

After the presence of genomic DNA had been confirmed by theelectrophoresis, the genomic DNA solution was 100 times diluted, and UVwas then measured with a spectrophotometer, so as to measure theconcentration of the genomic DNA. Based on the calculated concentration,the concentration of genomic DNA solution was adjusted to 10 ng/μl, andit was the used in the subsequent experiment.

(1-2) PCR

PCR was carried out according to a known method (Sambrook, J. D., W.Russell, Molecular Cloning: a Laboratory Manual, 3rd edn. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

A reaction solution having the following composition was prepared in a0.5-ml assist PCR tube.

Template (genomic DNA (10 ng)) 1.0 μl Primer (1) (10 pmol/μl) 1.0 μlPrimer (3) (10 pmol/μl) 1.0 μl 10 × Ex Taq buffer (Mg²⁺ plus) 2.5 μl(Mg²⁺ concentration: 20 mM) dNTP Mixture (2.5 mM each) 2.0 μl DMSO 1.25μl  Distilled water 16.1 μl  TaKaRa Ex Taq (5 U/μl) 0.15 μl  Totalvolume 25.0 μl 

15 μl of mineral oil was laminated on this reaction solution.Thereafter, the reaction was carried out using PTC-100™ ProgrammableThermal Controller.

The following primers were used.

Primer (3) ATP-NA1 Bam (10 pmol/μl) (31 mer, GC-Cont.: 54.8%)(SEQ ID NO: 11) 5′-GGA TCC ATG GCC GCA ATT ACA TCA GCT ACC G-3′Primer (1) PYC6-C-term SacI (10 pmol/μl) (30 mer, GC-Cont.: 43.3%)(SEQ ID NO: 7) 5′-GGA GCT CTT ACC AAC CTT TTT CAG ATT GAG-3′Universal FITC Forward primer (M13 Fw primer) (2 pmol/μl) (SEQ ID NO: 9)5′-CGC CAG GGT TTT CCC AGT CAC GAC-3′Universal FITC Reverse primer (M13 Rv primer) (2 pmol/μl)(SEQ ID NO: 10) 5′-GAG CGG ATA ACA ATT TCA CAC AGG-3′

The reaction was carried out under conditions of (94° C.-48 seconds, 64°C.-48 seconds, and 72° C.-1 minute 24 seconds)×31 cycles.

After completion of the reaction, confirmation was carried out by 2%(w/v) agarose gel electrophoresis. 2 μl of a gel loading buffer wasadded to 10 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TBE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera.

(1-3) Gel Extraction of PCR Product

A band that was considered to be DNA of interest was cut out of 2% (w/v)agarose gel, and it was then minced as finely as possible. The gel wasplaced in Amicon Ultra free (registered trade mark) DA, and it was thencentrifuged at 7,300 rpm at 4° C. for 10 minutes. After completion ofthe centrifugation, Ultra free MC (a column portion on the upper side)was removed, and to the eluted solution, an equal amount of TE-saturatedPhenol was added. Thereafter, the mixture was fully stirred using avortex, followed by centrifugation at 14,000 rpm at 4° C. for 10minutes. The water layer was transferred into a new 1.5 mlmicrocentrifuge tube, and 1/20 vol. of 3 M NaOAc (pH 5.2) and 2 vol. of99.5% ethanol were then added thereto. After the mixture had been fullystirred, it was left at −20° C. for 30 minutes. Thereafter, theresultant was centrifuged at 14,000 rpm at 4° C. for 10 minutes, and thesupernatant was then discarded. 500 μl of 75% ethanol was added to theprecipitate, and the precipitate was then fully washed. The resultantwas further centrifuged at 14,000 rpm at 4° C. for 5 minutes, and thesupernatant was then discarded. The precipitate was air-dried to acertain extent, and it was then dissolved in 20 μl of TE. In order toconfirm that the DNA of interest had been uniquely extracted andpurified in this solution, 2% (w/v) agarose gel electrophoresis wascarried out. 2 μl of a gel loading buffer was added to 10 μl of theobtained sample, and electrophoresis was carried out using anelectrophoresis buffer (1×TBE) at 100 V for 30 minutes. After completionof the electrophoresis, the gel was stained with 0.1 mg/ml ethidiumbromide for 7 minutes, and it was then decolorized with extra pure waterfor 10 minutes. The decolorized gel was placed on a UV transilluminator,and it was then photographed with a nonautomatic hooded instant camera.

(1-4) Subcloning of PCR Product

The subcloning of the PCR product was carried out according to a knownmethod (Hanahan, D., J. Mol. Biol., 166(4), 557-580 (1983); and HirokiNakayama et al., Bio Illustrated II, Idenshi Kaiseki no Kiso (Basic GeneAnalyses), Shujunsha Co., Ltd., 83-88 (1996)).

The PCR product and other components were prepared to have the followingcomposition.

Insert:Vector Insert:Vector (1:1) (3:1) PCR products 0.5 μl 1.5 μl pGEM(registered trade mark)-T 0.5 μl 0.5 μl Easy Vector 2 × Lapid LigationBuffer 5.0 μl 5.0 μl T₄ DNA Ligase 1.0 μl 1.0 μl Distilled water 3.0 μl3.0 μl Total volume 10.0 μl  10.0 μl 

The prepared reaction solution was incubated in a low temperatureincubator (16° C.) overnight for a ligation reaction. Escherichia coliwas transformed as follows. First, 150 μl of competent cells (DH5α) weremelted on ice to a certain extent, and 4 μl of the ligated reactionsolution was then gently added thereto. The mixture was softly stirredwith the tip of a chip, and it was then left on ice for 30 minutes.After it had left for 30 minutes, it was heat shocked at 42° C. for 20seconds. Thereafter, the resultant was left at rest on ice for 3minutes. Thereafter, each of 100 μl of and 50 μl of cell mass solutions(Vector:Insert=1:1, and 1:3) was applied on the entire surface of anLB-Ampicillin-X-Gal-IPTG plate, using a cell inoculation loop, and itwas then cultured at 37° C. overnight.

(1-5) Preparation of Plasmid According to Alkaline SDS Method

In this section, a plasmid was prepared in the same manner as thatdescribed in Example 1 (2-4).

(1-6) Preparation of Plasmid using QIAGEN Spin Miniprep Kit

In this section, a plasmid was prepared in the same manner as thatdescribed in Example 1 (2-5).

(1-7) Determination of Nucleotide Sequence according to Dideoxy Method

In this section, a nucleotide sequence was determined by the same methodas that described in Example 1 (2-6). The following results wereobtained.

High purity genomic DNA was extracted from 2 or 3 leaves of transgenicA. thaliana, using ISOPLANT II. In order to confirm the presence orabsence of introduction of a cytochrome c₆ gene used to be introducedinto plants, using the genomic DNA solution as a template, a gene ofinterest was amplified by PCR. Thereafter, the PCR product was subjectedto 2% (w/v) agarose gel (TBE) electrophoresis. As a result, a singleband was confirmed around 474 by (FIG. 9). In order to determine thenucleotide sequence of the amplified gene, after gel extraction,subcloning was carried out. Thereafter, the amplified gene was sequencedby the dideoxy method, so as to determine the entire nucleotide sequenceconsisting of 474 bp. The sequence results were compared with the knownsp gene sequence and PYC6 gene sequence, using GENETYX-MAC. As a result,as with the introduced gene, the determined nucleotide sequence wascompletely identical to those of the above genes with the exception thatT (thymine) at position 103 in the signal peptide gene sequence wassubstituted with A (adenine). From the above results, it was revealedthat an sp+PYC6 gene had been introduced into the plant body (A.thaliana).

(2) Analysis of Expression Gene in Total RNA according to RT-PCR Method

In the present section, total RNA was extracted from transgenicArabidopsis thaliana, and RT-PCR was carried out using the total RNA asa template. Thereafter, based on the presence or absence ofamplification of the introduced gene, expression of the gene in theplant body was confirmed.

Primer (3) ATP-NA1 Bam (10 pmol/μl) (31 mer, GC-Cont.: 54.8%)(SEQ ID NO: 11) 5′-GGA TCC ATG GCC GCA ATT ACA TCA GCT ACC G-3′Primer (1) PYC6-C-term SacI (10 pmol/μl) (30 mer, GC-Cont.: 43.3%)(SEQ ID NO: 7) 5′-GGA GCT CTT ACC AAC CTT TTT CAG ATT GAG-3′Universal FITC Forward primer (M13 Fw primer) (2 pmol/μl) (SEQ ID NO: 9)5′-CGC CAG GGT TTT CCC AGT CAC GAC-3′Universal FITC Reverse primer (M13 Rv primer) (2 pmol/μl)(SEQ ID NO: 10) 5′-GAG CGG ATA ACA ATT TCA CAC AGG-3′(2-1) Extraction of Total RNA from Transformant

Total RNA was extracted from a transformant using a commerciallyavailable kit (RNeasy Plant Mini Kit (QIAGEN)) in accordance with aninstruction included therewith.

(2-2) PCR

PCR was carried out according to a known method (Sambrook, J. D., W.Russell, Molecular Cloning: a Laboratory Manual, 3rd edn. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

A reaction solution having the following composition was prepared in a0.5-ml assist PCR tube.

Template (total RNA (10 ng)) 1.0 μl Primer (1) (10 pmol/μl) 1.0 μlPrimer (3) (10 pmol/μl) 1.0 μl 10 × Ex Taq buffer (Mg²⁺ plus) 2.5 μl(Mg²⁺ concentration: 20 mM) dNTP Mixture (2.5 mM each) 2.0 μl DMSO 1.25μl  Distilled water 16.1 μl  TaKaRa Ex Taq (5 U/μl) 0.15 μl  Totalvolume 25.0 μl 

15 μl of mineral oil was laminated on the above reaction solution.Thereafter, a reaction was carried out using PTC-100™ ProgrammableThermal Controller.

After completion of the reaction, confirmation was carried out by 2%(w/v) agarose gel electrophoresis. 2 μl of a gel loading buffer wasadded to 10 μl of the obtained PCR product, and the obtained mixture wasused as an electrophoretic sample. Electrophoresis was carried out at100 V for 30 minutes using an electrophoresis buffer (1×TBE). Aftercompletion of the electrophoresis, the gel was stained with 0.1 mg/mlethidium bromide for 7 minutes, and it was then decolorized with extrapure water for 10 minutes. The decolorized gel was placed on a UVtransilluminator, and it was then photographed with a nonautomatichooded instant camera.

(2-3) Gel Extraction of PCR Product

In this section, gel extraction of the PCR product was carried out inthe same manner as that described in Example 3 (1-3).

(2-4) Subcloning of PCR Product

In this section, the subcloning of the PCR product was carried out bythe same method as that described in Example 3 (1-4).

(2-5) Preparation of Plasmid according to Alkaline SDS Method

In this section, the alkaline SDS method was carried out according toknown means by the same method as that described in Example 1 (2-4)(Weiss. B, et al., J. Biol. Chem., 243, 4543-4555 (1968); Birnboim, H.C., Methods Enzymol., 100, 243 (1983)).

(2-6) Preparation of Plasmid using QIAGEN Spin Miniprep Kit

In this section, a plasmid was prepared using QIAGEN Spin Miniprep Kitby the same method as that described in Example 1 (2-5).

(2-7) Determination of Nucleotide Sequence According to Dideoxy Method

In this section, a nucleotide sequence was determined by the same methodas that described in Example 1 (2-6) (Sanger, F., Sanger, F.,Determination of nucleotide sequence in DNA., Science, 214, 1205-1210(1981)).

(2-8) Results

The expressed total RNA was extracted from 2 or 3 leaves of a transgenicplant (PYC6-introduced plant) wherein introduction of the gene had beenconfirmed, using RNeasy Plant Mini Kit. In order to confirm the presenceor absence of expression of an sp+PYC6 gene, using the total RNAsolution as a template, a gene of interest was amplified by RT-PCR.Thereafter, the PCR product was subjected to 2% (w/v) agarose gel (TBE)electrophoresis. As a result, a single band was confirmed around 474 by(FIG. 9). In order to determine the nucleotide sequence of the amplifiedgene, after gel extraction, subcloning was carried out. Thereafter, theamplified gene was sequenced by the dideoxy method, so as to determinethe entire nucleotide sequence consisting of 474 bp. The sequenceresults were compared with the known sp gene sequence and PYC6 genesequence, using GENETYX-MAC. As a result, as with the introduced gene,the determined nucleotide sequence was completely identical to those ofthe above genes with the exception that T (thymine) at position 103 inthe signal peptide gene sequence was substituted with A (adenine). Fromthe above results, it was considered that the sp+PYC6 gene had beenexpressed in the plant body (A. thaliana).

(3) Analysis of Expressed Protein According to Western Blotting Method

In this section, chloroplast was extracted from transgenic Arabidopsisthaliana. Thereafter, it was confirmed that the introduced cytochrome c₆had been expressed in a chloroplast protein fraction obtained from theextracted chloroplast.

(3-1) Preparation of Chloroplast Fraction

A chloroplast fraction was prepared according to known means (KenzoNakamura et al., (edited), Shokubutsu no Tanpakushitsu Jikken Protocol(Plant Protein Experimental Protocols), Saibo Kogaku Series (CellTechnology Series), Vol. 9, Shujunsha Co., Ltd., 114-117 (1998)).

Approximately 5 to 6 g of transgenic Arabidopsis thaliana was placed ina mortar that had previously been cooled, and liquid nitrogen wasrapidly added thereto. Thereafter, the plant body was homogenized untilit became a powder state. Thereafter, 20 ml of disintegration bufferthat had been cooled on ice was added to the powder sample, and themixture was then fully stirred. Thereafter, the mixture was filtratedwith 4 layers of Miracloth. The obtained filtrate was transferred into a15-ml corning tube, and it was then centrifuged at 7,000 rpm at 4° C.for 15 seconds. Thereafter, the supernatant was carefully eliminated. 5vol. of disintegration buffer was added to the precipitate, and theywere blended so as to form a homogeneous solution. Thereafter, theobtained solution was gently laminated on a glass centrifuge tube, inwhich 10 ml of 30% Percoll had previously been placed. It was thencentrifuged at 2,000 rpm at 4° C. for 3 minutes. After completion of thecentrifugation, Percoll as a supernatant was eliminated, and theprecipitate was mixed into 5 ml of disintegration buffer, until theobtained mixture became homogeneous. The mixture was centrifuged at2,000 rpm, at 4° C. for 3 minutes, again. (This operation was repeatedtwice). Finally, the precipitate was dissolved in 1 ml of disintegrationbuffer, and the solution was defined as a chloroplast fraction.

(3-2) Western Blotting

1 ml of CelLytic P Plant Cell Lysis/Extraction reagent was added to thechloroplast fraction solution obtained in (3-1) above, and the obtainedmixture was then centrifuged at 10,000 rpm at 4° C. for 10 minutes. Theobtained supernatant was defined as a chloroplast protein extractionsolution. 5 μl of a 1 μg/μl molecular weight standard marker, 5 μl ofextra pure water, and the chloroplast protein extraction solution wereadded to a 1.5 ml microcentrifuge tube. To each tube, an equal amount ofsample buffer was added, and the mixture was then fully stirred. Theprepared tube was incubated at 40° C. for 30 minutes in a hot-waterbath, and it was then used as an electrophoretic sample.

An anode electrode solution, a cathode electrode solution, and SDS-PAGEgel were set in an electrophoretic tank, and 20 μl of theelectrophoretic sample was carefully applied in a well. Thereafter, theelectrophoretic sample was electrophoresed up to the upper end ofresolving gel at a constant voltage of 30 V, and the marker waselectrophoresed up to approximately 5 mm from the lower end of the gelat a constant voltage of 100 V. During the electrophoresis, two filterswere immersed in each of blotting buffers A, B, and C. The PVDF membranewas immersed in a small amount of methanol for 5 seconds and was thenimmersed in the blotting C solution. After completion of theelectrophoresis, the gel was removed from the glass board, and it wasthen immersed in the blotting C solution for approximately 5 minutes.Two filters A, two filters B, the PVDF membrane, the gel, and twofilters C were laminated in this order from the bottom of a semi-dryblotting device. After they had been laminated, transcription wasconducted at a constant current of 60 mA for 90 minutes. Immediatelyafter completion of the blotting, operations were conducted in thefollowing orders, so as to carry out an antigen-antibody reaction.

(i) The PVDF membrane is immersed in TBS and is then shaken for 5minutes (2 times);

(ii) It is immersed in 5% BSA in TBS and is then shaken from 1 hour toovernight;

(iii) It is immersed in TBS, and is then shaken for 5 minutes;

(iv) It is immersed in 1^(st)-antibody, and is then shaken for 2 hours;

(v) It is immersed in TTBS, and is then shaken for 5 minutes (2 times);

(vi) It is immersed in 2^(nd)-antibody, and is then shaken for 1 hour 30minutes;

(vii) It is immersed in TTBS, and is then shaken for 5 minutes (2times);

(viii) It is immersed in TBS, and is then shaken for 5 minutes (2times);

(ix) It is immersed in a 2″-antibody development reagent, and is thenshaken fro 20 minutes (light-blocked); and

(x) It is washed with extra pure water.

(3-3) Results

A chloroplast fraction was separated from a PYC6-introduced plant byPercoll density gradient centrifugation. The obtained chloroplastproteins were electrophorestically screened by SDS-PAGE in terms ofmolecular weight. Subsequently, all of the separated proteins weretranscribed onto a PVDF membrane, and an antigen-antibody reaction wascarried out using a primary antibody (rabbit antibody) and ananti-rabbit antibody (secondary antibody) that recognized cytochrome c₆derived from P. yezoensis. As a result, such an antigen-antibodyreaction could be detected only in the PYC6-introduced plant (FIG. 10).From such results, it was revealed that PYC6 introduced into thePYC6-introduced plant was expressed in the chloroplast.

(4) Analysis of N-Terminal Amino Acid Sequence of Expressed Cytochromec₆ Protein

In this section, the N-terminal amino acid sequence of a PYC6 proteinthat exhibited an antigen-antibody reaction in (3) above was analyzed,and the presence or absence of the cleavage of a signal peptide was thenconfirmed.

1 ml of CelLytic P Plant Cell Lysis/Extraction reagent was added to thechloroplast fraction solution obtained in (3-1) above, and the obtainedmixture was then centrifuged at 10,000 rpm at 4° C. for 10 minutes. Theobtained supernatant was defined as a chloroplast protein extractionsolution. 5 μl of a 1 μg/μl molecular weight standard marker, 5 μl ofextra pure water, and the chloroplast protein extraction solution wereadded to a 1.5 ml microcentrifuge tube. To each tube, an equal amount ofsample buffer was added, and the mixture was then fully stirred. Theprepared tube was incubated at 40° C. for 30 minutes in a hot-waterbath, and it was then used as an electrophoretic sample.

An anode electrode solution, a cathode electrode solution, and SDS-PAGEgel were set in an electrophoretic tank, and 20 μl of theelectrophoretic sample was carefully applied in a well. Thereafter, theelectrophoretic sample was electrophoresed up to the upper end ofresolving gel at a constant voltage of 30 V, and the marker waselectrophoresed up to approximately 5 mm from the lower end of the gelat a constant voltage of 100 V. During the electrophoresis, two filterswere immersed in each of blotting buffers A, B, and C. The PVDF membranewas immersed in a small amount of methanol for 5 seconds and was thenimmersed in the blotting C solution. After completion of theelectrophoresis, the gel was removed from the glass board, and it wasthen immersed in the blotting C solution for approximately 5 minutes.Two filters A, two filters B, the PVDF membrane, the gel, and twofilters C were laminated in this order from the bottom of a semi-dryblotting device. After they had been laminated, transcription wasconducted at a constant current of 60 mA for 90 minutes. Aftercompletion of the blotting, CBB staining and discoloration operationwere conducted, and a protein portion of interest was cut into a size of1 mm×5 mm, and the thus cut protein portion was then transferred into a1.5 ml microcentrifuge tube. This was used as a sequence sample, and theamino acid sequence thereof was analyzed using Applied BiosystemsProtein Sequencer 492.

The N-terminal amino acid sequence of a protein with a molecular weightof approximately 9.1 kDa having an antigen-antibody reaction with a PYC6antibody obtained from the transgenic plant was analyzed by the Edmanmethod. As a result, the sequence was found to be ADLDNGEKVF (SEQ ID NO:12) from the 1^(st) residue on the N-terminus (please refer to thefollowing table).

Molecular N-terminal mass (kDa) sequence P. yezoensis cyt c₆ 9.1ADLDNGEKVF PYC6 9.1 ADLDNGEKVF

This sequence is completely identical to the N-terminal amino acidsequence of the mature protein region of PYC6. From this result, it wasrevealed that the signal peptide had been cleaved in the plant body, andthat PYC6 precisely had passed through the chloroplast thylakoidmembrane and had been expressed on the lumen side. That is to say, as aresult of the analysis of the N-terminal amino acid sequence of the PYC6protein in the obtained transformant, it was found that the abovesequence was identical to the amino acid sequence of the mature regionwherein a chloroplast transport signal had been cleaved. Thus, it couldbe confirmed that the sp+PYC6 gene was transcribed and translated, andwas then transported to the chloroplast.

Example 4 Measurement of Growth of Transgenic Plant and Observation ofPhenotype (1) Growth Observation (Height, Root Length, and Leaf Size)

In this section, the growth of transgenic Arabidopsis thaliana afterinsemination was observed.

After the transgenic plant had been inseminated, the “height,” “rootlength,” and “leaf size” of the plant body were observed and measuredevery 10 days. As plant samples, 20 individual wild-type strains and 20individual transgenic plants were prepared. The mean value wascalculated, and a significant difference was then obtained bystatistical processing.

As a result of observation of the growth (phenotype) of thePYC6-introduced plants, the height of such a plant was at maximum 1.9times greater than that of the wild-type plant (WT) (40 days afterinsemination), and it was then 1.3 times greater than that of WT on theapproximately 60^(th) day (FIG. 11). The root length of such aPYC6-introduced plant was at maximum 1.35 times greater than that of WT(40 days after insemination), and it was then 1.17 times greater thanthat of WT on the approximately 60^(th) day (FIG. 12). The leaf size ofsuch a PYC6-introduced plant was at maximum 1.9 times greater than thatof WT (40 days after insemination), and it was then 1.3 times greaterthan that of WT on the approximately 60^(th) day (FIG. 13). It wasconsidered that such phenomena occurred in such PYC6-introduced plantswere brought on due to activation of a photosynthetic reaction extremelyimportant for the growth of plants as a result of expression of PYC6 inthe plant bodies. Thus, it was considered that the growth rate of theplant bodies was increased by the influence of such activation of aphotosynthetic reaction.

(2) Measurement of Total Chlorophyll Amount

In this section, the total amount of chlorophyll was extracted fromtransgenic Arabidopsis thaliana using acetone, and it was thenquantified.

Leaves of transgenic Arabidopsis thaliana were collected using a leafpunch, and they were then weighed. The sample was placed in a mortarthat had previously been cooled, and liquid nitrogen was then rapidlyadded thereto. The mixture was homogenized until the plant body became apowder state. Thereafter, 80% acetone was added to the powder sample, soas to adjust the volume to 4 ml. The absorbance values at 663 nm and at645 nm of the above solution were measured using a spectrophotometerUV-VISIBLE SPECTROPHOTOMETER.

The obtained absorbance values at 663 nm and at 645 nm were assignedinto the following formula, so as to calculate the chlorophyll amount.

Total chlorophyll amount (μg/ml)=8.02×ABS(663)+20.21×ABS(645)

As a result of the measurement of the chlorophyll amount in thePYC6-introduced plant, it was found that the chlorophyll amount in thePYC6-introduced plant was 1.1 to 1.2 times larger than that of WT duringthe period from 40 to 70 days after insemination (FIG. 14). It wasconsidered that this was because a light reaction was activated byintroduction of PYC6, and because an energetic substance (ATP) that wasthe thus increased final product of the light reaction was utilized inthe synthesis of chlorophyll.

Example 5 Measurement of Photosynthetic Activity of Transgenic Plant (1)Methods and Results

In order to use as indicators of photosynthetic activity, an adeninenucleotide (ATP) content, an NADPH amount, carbon dioxide-assimilatingability, a starch amount, and a protein amount were measured by thefollowing methods.

(1-1) Measurement of Total ATP Content

In the present example, total ATP was extracted from transgenicArabidopsis thaliana, and it was then quantified by theluciferin-luciferase method.

Leaves of transgenic Arabidopsis thaliana had been weighed in advance.After weighing, the sample was placed in a mortar that had previouslybeen cooled, and liquid nitrogen was then rapidly added thereto. Themixture was homogenized using a pestle until the plant body became apowder state. Thereafter, 2 ml of 0.25 M perchloric acid was added tothe powder sample, and the obtained solution was then recovered in a15-ml corning tube. The solution was centrifuged at 10,000 rpm at 4° C.for 10 minutes. Thereafter, the obtained supernatant was transferredinto another 15-ml corning tube, and the pH was then adjusted to pH 7.0with 1 N KOH. After the pH had been adjusted, the volume was adjusted to5 ml with a 0.2 M phosphate buffer (pH 7.2), and the thus obtainedsolution was defined as an ATP extract.

100 μl of the ATP extract was prepared in a 1.5 ml microcentrifuge tube,and 20 μl of ENLITEN Luciferase/Luciferin Reagent was added thereto. 90seconds after addition of the above agent, the integral of theluminescence intensity of the reaction solution was calculated for 10seconds using a luminometer (GENE LIGHT 55). Thereafter, the ATPconcentration in the solution was calculated from the obtained RLUvalue, and the ATP amount per weight of the plant was obtained from theobtained ATP concentration.

As a result, the total amount of ATP contained in the PYC6-introducedplant was increased by at maximum 1.93 times (60 days afterinsemination) (FIG. 15). It was considered that this was because P.yezoensis cyt c₆(PYC6) functioned as a new electron carrier, as well asplastocyanin functioning in the PYC6-introduced plant, so that electrontransfer in a light reaction could be activated, and so that the amountof ATP as a final product was thereby increased.

(1-2) Measurement of Total NADPH Content

In the present example, total NADPH was extracted from transgenicArabidopsis thaliana, and it was then quantified.

Transgenic Arabidopsis thaliana had been weighed in advance. Thereafter,0.1 N NaOH was placed in the weighed transgenic plant, which had beenheated with a hot bath at approximately 70° C., and the mixture was thendisintegrated with a Polytron homogenizer. Thereafter, the resultant wastransferred into an ice bath, and approximately 2 ml of 0.1 N HCl wasadded thereto. The obtained mixture was adjusted to pH 7.5 using a pHtest paper. Subsequently, 0.1 ml of glycylglycine buffer (pH 7.5) wasadded to the mixture, and the obtained mixture was then fully stirred.Thereafter, the reaction solution was transferred into a measuringcylinder, and the liquid amount was then measured. After completion ofthe measurement, the solution was transferred into a 15-ml corning tube,and it was then centrifuged at 10,000 rpm at 4° C. for 20 minutes. Aftercompletion of the centrifugation, the supernatant was recovered in a15-ml corning tube, and it was then cooled at −80° C. overnight. Afterthe resultant had been dissolved, it was then centrifuged at 10,000 rpmat 4° C. for 20 minutes, and the resultant was then used inquantification.

100.0 μl of an NADPH extract, 346.0 μl of a 0.1 M glycylglycine buffer(pH 7.4), 200.0 μl of 0.2 M nicotinamide, 82.0 μl of 3.2 mg/ml phenazinemethosulfate (PMS), 67.0 μl of 5 mg/ml thiazole blue, and 200 μl ofglucose-6-dibasic sodium phosphate hydrate, were blended. The obtainedmixture was set in a spectrophotometer U3310. Measurement was carriedout at 570 nm at 25° C. A temperature control unit was installed inU3310, and the temperature thereof was set at 25° C. Thereafter, achange in the absorbance at 570 nm was measured every 30 seconds for 2minutes, and based on differences in the obtained absorbance values, theamount of NADPH was calculated. Thereafter the amount of NADPH perweight of the plant was calculated from the above obtained value.

As a result, the total amount of NADPH contained in the PYC6-introducedplant was increased by 1.2 to 1.4 times on 40 to 70 days afterinsemination (FIG. 16). It was considered that this was because largerquantities of electrons were transferred into the PYC6-introduced higherplant than into a wild-type plant as a result of introduction of PYC6into the higher plant, and because the amount of NADPH as a finalproduct was thereby increased.

(1-3) Measurement of Carbon Dioxide-Assimilating Ability

In the present example, using leaves of transgenic Arabidopsis thaliana,carbon dioxide-assimilating ability was measured.

Such carbon dioxide-assimilating ability was measured using a CO₂ gasanalyzer CIRAS-1 (manufactured by Koito Industries, Ltd.). In addition,the amount of carbon dioxide supplied was set at 350 ppm, and themeasurement was then carried out under saturated light.

As a result, the carbon dioxide-assimilating ability of thePYC6-introduced plant was increased by at maximum 2.2 times (50 daysafter insemination) (FIG. 17). It was considered that this was becausethe amount of ATP and the amount of NADPH were increased by introductionof PYC6 into a higher plant, and a dark reaction (Calvin-Benson cycle)that utilized such energy was thereby activated, so that the carbondioxide-assimilating ability was increased.

(1-4) Measurement of Starch Amount

In the present example, starch was extracted from transgenic Arabidopsisthaliana, and it was then quantified.

Plant leaves were collected and were then dried (70° C., 2 days).Approximately 5 mg of leaves were weighed out, and they were then placedin a mortar. Thereafter, 3 ml of 32% perchloric acid was added thereto,and the obtained mixture was then fully disintegrated with a pestle.Thereafter, all the obtained sample was placed in a test tube, and itwas then left at rest at 20° C. for 20 minutes. Subsequently, theresultant was filtrated with a 6.0-cm Whatman GF/A grass fibre disk.Thereafter, 5 ml of an iodine solution was then added to the filtrate,and they were then well blended. The obtained mixture was left at restat approximately 4° C. for 30 minutes. Thereafter, the resultant wasfiltrated with a 1.5-cm Whatman GF/A grass fibre disk, and the filterwas then dried.

The dried filter was placed in a test tube, and 4 ml of 0.75 M sulfuricacid was then added thereto. The obtained mixture was incubated in aboiled water bath for 30 minutes. Thereafter, the supernatant wasrecovered, and color was developed therefrom according to the phenolsulfuric acid method. Thereafter, the absorbance at 485 nm was measuredusing a spectrophotometer.

As a result, the amount of starch contained in the PYC6-introduced plantwas increased by 1.15 to 1.25 times for 40 to 70 days after insemination(FIG. 18). It was considered that this was because the CO₂-assimilatingability of the PYC6-introduced higher plant was increased byintroduction of PYC6 into the higher plant, and the synthesis of starchwas also promoted thereby.

(1-5) Measurement of Protein Amount

In the present example, protein was extracted from transgenicArabidopsis thaliana, and it was then quantified by the Lowry method.

The weight of a plant body was measured and recorded. A suitable amountof plant body was placed in a mortar, and it was then sufficientlycrushed with a pestle in the presence of liquid nitrogen. Thetemperature was returned to room temperature, and 2 ml of CelLytic P(manufactured by Sigma) was then added to 1 g of the plant body. Theobtained mixture was sufficiently crushed with a pestle, and theresultant was then transferred into a test tube. The test tube wasturned upside down at room temperature for 15 minutes, so that themixture could be blended, followed by centrifugation. The obtainedsupernatant was defined as an extracted protein.

Subsequently, the following solutions were produced: Lowry A solutionprepared by adding sodium carbonate to a 0.1 N sodium hydroxide solutionresulting in 2%; Lowry B solution containing 0.5% copper sulfatepentahydrate and 1% sodium citrate; Lowry C solution consisting of a 1 Nphenol solution; and Lowry D solution consisting of Lowry A solution andLowry B solution. Thereafter, 1.0 ml of the Lowry D solution was addedto 0.1 ml of the extracted protein solution, and the obtained mixturewas then stirred using a vortex mixer, followed by incubation at 30° C.for 15 minutes. Subsequently, 0.1 ml of the Lowry C solution was addedto the reaction solution, and the obtained mixture was rapidly stirredwith a vortex mixer. The resultant was incubated at 30° C. for 30minutes, and the absorbance at 770 nm was then measured.

As a result, the amount of protein contained in the PYC6-introducedplant was increased by at maximum 1.3 times (50 days after insemination)(FIG. 19). It was considered that this was because ATP and NADPHincreased by introduction of PYC6 into a higher plant activated thesynthesis of the protein.

(2) Consideration

By the present invention, it was found that the PYC6-introduced planthad a growth rate that was higher than that of WT. It was assumed thatsince ATP and NADPH contained in the PYC6-introduced plant wereincreased, P. yezoensis cyt c₆ acting as a newly introduced electroncarrier functioned together with plastocyanin functioning in A.thaliana, resulting in two cycles of electron transfer in a lightreaction. Accordingly, the amounts of ATP and NADPH as final productswere increased, so that the growth of the plant as a whole could beactivated. To date, an increase in the component amount of a land plantdue to the reinforcement of enzymes associated with a dark reaction(Calvin-Benson cycle) has been studied. However, there have been nocases of activating a light reaction.

Moreover, to date, there have been no cases of introducing cyt c₆ into aplant to allow it function therein. Thus, the contents of the presentinvention are sufficiently applicable to other types of plants in thefuture. Accordingly, it can be said that the present invention hasversatility and that it becomes a base for a method of promoting thegrowth of a useful plant, a plant production technique (fruit plants,foliage plants, real flowers, etc.), and the like. Furthermore, othertypes of metabolism are also activated utilizing ATP and NADPH increasedin a plant body, and thus it was considered that the present inventionwas extremely effective for the field of a method of eliminatingcontaminants (CO₂, etc.).

INDUSTRIAL APPLICABILITY

The present invention provides a method of producing a novel higherplant having cytochrome c₆ in the thylakoid space of chloroplast. Thepresent invention also provides a method of promoting the growth of ahigher plant, a method of promoting the synthesis of ATP, NADPH, astarch, and a protein, and a method of promoting carbon fixation.

Moreover, the present invention further provides a signal peptide-addedcytochrome c₆ protein that can be transported into the thylakoid spaceof chloroplast, a gene encoding the above protein, a recombinant vectorcomprising the above gene, a transformant comprising the aboverecombinant vector, and a transgenic higher plant which is obtained byintroducing the above gene into the plant genome thereof.

Sequence Listing Free Text SEQ ID NO: 7—Primer SEQ ID NO: 8—Primer SEQID NO: 9—Primer SEQ ID NO: 10—Primer

SEQ ID NO: 11—Primer

1. A method of producing a higher plant having cytochrome c₆ in thethylakoid space of chloroplast, comprising introducing a gene encoding afused protein comprising a signal peptide consisting of 50 to 80 aminoacid residues and a cytochrome c₆ protein into the genome of a higherplant, wherein the cytochrome c₆ protein is from Porphyra yezoensis. 2.A method of promoting the growth of a higher plant, comprisingintroducing a gene encoding a fused protein comprising a signal peptideconsisting of 50 to 80 amino acid residues and a cytochrome c₆ proteininto the genome of a higher plant, expressing the gene under suitableconditions, wherein the cytochrome c₆ is located in the thylakoid spaceof chloroplast, wherein the cytochrome c₆ protein is from Porphyrayezoensis.
 3. A method of promoting the synthesis of at least oneselected from the group consisting of the ATP, NADPH, starch, andprotein of a higher plant, comprising introducing a gene encoding afused protein comprising a signal peptide consisting of 50 to 80 aminoacid residues and a cytochrome c₆ protein into the genome of a higherplant, expressing the gene under suitable conditions, wherein thecytochrome c₆ is located in the thylakoid space of chloroplast, whereinthe cytochrome c₆ protein is from Porphyra yezoensis.
 4. A method ofpromoting carbon fixation by a higher plant, comprising introducing agene encoding a fused protein formed by adding a signal peptideconsisting of 50 to 80 amino acid residues and a cytochrome c₆ proteininto the genome of a higher plant, expressing the gene under suitableconditions, wherein the cytochrome c₆ is located in the thylakoid spaceof chloroplast, wherein the cytochrome c₆ protein is from Porphyrayezoensis.
 5. The method according to any one of claims 1 to 4, whereinsaid fused protein is the protein described in the following (a), (b),(c), or (d): (a) a protein having the amino acid sequence as shown inSEQ ID NO: 6; (b) a protein, which has an amino acid sequence comprisinga deletion, substitution, or addition of one or several amino acids withrespect to an amino acid sequence portion corresponding to said signalpeptide in the amino acid sequence as shown in SEQ ID NO: 6, and whichhas ability to pass through the chloroplast envelope and thylakoidmembrane of a higher plant; (c) a protein, which has an amino acidsequence comprising a deletion, substitution, or addition of one orseveral amino acids with respect to an amino acid sequence portioncorresponding to said cytochrome c₆ protein in the amino acid sequenceas shown in SEQ ID NO: 6, and which has ability to transfer electrons;and (d) a protein, which has an amino acid sequence comprising adeletion, substitution, or addition of one or several amino acids withrespect to each of an amino acid sequence portion corresponding to saidsignal peptide and an amino acid sequence portion corresponding to saidcytochrome c₆ protein in the amino acid sequence as shown in SEQ ID NO:6, and which has ability to pass through the chloroplast envelope andthylakoid membrane of a higher plant and ability to transfer electrons.6. The method according to any one of claims 1 to 4, wherein said geneis a gene comprising the DNA described in the following (a) or (b): (a)DNA having the nucleotide sequence as shown in SEQ ID NO: 5; and (b)DNA, which hybridizes with DNA having a nucleotide sequencecomplementary to the DNA having the nucleotide sequence as shown in SEQID NO: 5 under stringent conditions, and which encodes a protein havingability to pass through the chloroplast envelope and thylakoid membraneof a higher plant and ability to transfer electrons.
 7. A fused protein,comprising a signal peptide consisting of 50 to 80 amino acid residuesand a cytochrome c₆ protein, wherein the cytochrome c₆ protein is fromPorphyra yezoensis.
 8. The fused protein according to claim 7, which isthe protein described in the following (a), (b), (c), or (d): (a) aprotein having the amino acid sequence as shown in SEQ ID NO: 6; (b) aprotein, which has an amino acid sequence comprising a deletion,substitution, or addition of one or several amino acids with respect toan amino acid sequence portion corresponding to said signal peptide inthe amino acid sequence as shown in SEQ ID NO: 6, and which has abilityto pass through the chloroplast envelope and thylakoid membrane of ahigher plant; (c) a protein, which has an amino acid sequence comprisinga deletion, substitution, or addition of one or several amino acids withrespect to an amino acid sequence portion corresponding to saidcytochrome c₆ protein in the amino acid sequence as shown in SEQ ID NO:6, and which has ability to transfer electrons; and (d) a protein, whichhas an amino acid sequence comprising a deletion, substitution, oraddition of one or several amino acids with respect to each of an aminoacid sequence portion corresponding to said signal peptide and an aminoacid sequence portion corresponding to said cytochrome c₆ protein in theamino acid sequence as shown in SEQ ID NO: 6, and which has ability topass through the chloroplast envelope and thylakoid membrane of a higherplant and ability to transfer electrons.
 9. A gene, which encodes thefused protein according to claim 7 or
 8. 10. A gene, which comprises theDNA described in the following (a) or (b): (a) DNA having the nucleotidesequence as shown in SEQ ID NO: 5; and (b) DNA, which hybridizes withDNA having a nucleotide sequence complementary to the DNA having thenucleotide sequence as shown in SEQ ID NO: 5 under stringent conditions,and which encodes a protein having ability to pass through thechloroplast envelope and thylakoid membrane of a higher plant andability to transfer electrons.
 11. A recombinant vector comprising thegene according to claim
 9. 12. A transformant comprising the recombinantvector according to claim
 11. 13. The transformant according to claim12, wherein the transformant is a microorganism belonging to genusAgrobacterium.
 14. A transgenic higher plant, into the plant genome ofwhich the gene according to claim 9 has been introduced.
 15. Thetransgenic higher plant according to claim 14, which has cytochrome c₆in the thylakoid space of chloroplast in a plant cell thereof.
 16. Themethod according to any one of claims 1 to 4, wherein the higher plantbelongs to a plant family selected from the group consisting ofsolanaceae, gramineae, leguminosae, chenopodiaceae, rosaceae,asteraceae, liliaceae, caryophyllaceae, cucurbitaceae, convolvulaceae,amaranthaceae, bromeliaceae, cactaceae, aloeaceae and orchidaceae. 17.The method according to any one of claims 1 to 4, wherein the higherplant is a C4 plant, a Crassulaean acid metabolism plant, or a C3-C4intermediate plant.
 18. The method according to any one of claims 1 to4, wherein the higher plant is a C3 plant selected from the groupconsisting of solanaceae, gramineae, leguminosae, chenopodiaceae,rosaceae, asteraceae, cucurbitaceae, convolvulaceae and orchidaceae. 19.The transformant according to claim 12, wherein the transformant isselected from the group consisting of Escherichia coli, Bacillussubtilis, a yeast cell and a mold cell.